Factor VIIa inhibitors from Kunitz domain proteins

ABSTRACT

A potent serine protease inhibitor capable of inhibiting Factor VIIa, Factor XIa, plasma kallikrein, or plasmin is provided. The inhibitor is provided in a pharmaceutical composition for treatment of diseases where inhibition of Factor VIIa, Factor XIa, plasma kallikrein, or plasmin is indicated.

This application is a continuation-in-part of co-pending U.S.application Ser. No. 08/206,310 filed 4 Mar. 1994.

FIELD OF THE INVENTION

This invention relates to novel Kunitz domain proteins having tissuefactor-Factor VIIa inhibiting activity, DNA encoding these proteins, andrecombinant materials and methods for producing these Factor VIIainhibitors. The invention further relates to pharmaceutical compositionscontaining Factor VIIa inhibitors for treatment of diseases whereinhibition of Factor VIIa, Factor XIa, plasma kallikrein, and plasmin isindicated.

BACKGROUND OF THE INVENTION

Thrombosis accounts for about 40% of the deaths in the United States.Current treatments for thrombotic disorders involve the use ofanticoagulant drugs (e.g., heparin, coumadin) that have non-specificmechanisms of action. These drugs can cause bleeding thus limiting theiruse. Anticoagulants that block early steps in the coagulation cascade,and are specific for either the intrinsic or extrinsic pathways (seeFIG. 1), could have superior efficacy and safety profiles becausethrombotic events are suppressed without inducing bleeding episodes.

Factor VIIa

The tissue factor-Factor VIIa complex (TF-FVIIa) is a primary initiatorof blood coagulation (Carson, S. D. and Brozna, J. P., Blood Coag.Fibrinol. 4: 281-292 1993!; Davie, E. W. et al., Biochemistry 30:10363-10370 1991!; Rapaport, S. I. and L. V. M. Rao, Arterioscler.Thromb. 12: 1111-1121 1992!) (see FIG. 1). Factor VIIa (FVIIa), a 50kDa, vitamin K-dependent, plasma serine protease, is generated byproteolysis of a single peptide bond from its zymogen Factor VII (FVII),which is present at ca. 0.5 μg/ml in plasma. Tissue factor (TF) contains263 residues and is an integral membrane cofactor that is expressedconstitutively in cells separated from plasma by the vascularendothelium (Carson, S. D. and J. P. Brozna, Blood Coag Fibrinol 4:281-292 1993!). Upon tissue injury, the exposed extracellular domain ofTF can bind and activate FVII to form a high affinity TF-FVIIa complex.Factor XIIa (FXIIa) has also been implicated in the activation of FVII.The TF-FVIIa complex initiates the extrinsic pathway of the coagulationcascade by activation of Factor X (FX) to Factor Xa (FXa), Factor IX(FIX) to Factor IXa (FIXa), and additional FVII to FVIIa. This leads tothe conversion of prothrombin to thrombin, which carries out manybiological functions (Badimon, L. et al., Trends Cardiovasc. Med. 1:261-267 1991!). Among the most important functions of thrombin is theconversion of fibrinogen to fibrin, which polymerizes to form a clot.

The regulation of coagulation is critical to maintaining hemostasis.Following initiation of the coagulation cascade, TF-FVIIa is regulatedby tissue factor pathway inhibitor (TFPI), a feedback inhibitor thatprevents further activation of zymogen substrates (Broze Jr., G. J. etal., Biochemistry 29: 7539-7546 1990!; Broze Jr., G. J., Semin. Hematol.29: 159-169 1992!). TFPI is also known as LACI or EPI for lipoproteinassociated coagulation inhibitor and extrinsic pathway inhibitor,respectively. TFPI contains an acidic amino terminal region followed bythree Kunitz-type domains and a basic carboxy terminal region. TFPI isthought to inhibit TF-FVIIa in a FXa dependent manner, first binding FXavia the second Kunitz domain followed by binding FVIIa via the firstKunitz domain (Girard, T. J. et al., Nature 338: 518-520 1989!). In theabsence of FXa, TFPI is a poor inhibitor of the TF-FVIIa complex(Girard, T. J. et al., Science 248: 1421-1424 1990!). Recently, theserpin antithrombin III (AT III) has also been shown to inhibit TF-FVIIaactivity in the presence of heparin (Rao, L. V. M. et al., Blood 81:2600-2607 1993!; Lawson, J. H. et al., J. Biol. Chem. 268: 767-7701993!; Broze Jr., G. J. et al., Blood 82: 1679-1680 1993!; Mann, K. G.,Blood 82: 1680-1681 1993!). Inhibition of TF-FVIIa by TFPI isreversible, whereas inhibition by ATIII is essentially irreversible. Therelative importance of ATIII/heparin inhibition of TF-FVIIa versus TFPIis unknown in vivo.

Variants of TFPI have been made that also inhibit TF-FVIIa activity. Inparticular a variant that contains the first two Kunitz domains(residues 1-161) has been made and characterized (Hamamoto et al. J.Biol. Chem. 268: 8704-8710 1993!; Petersen et al. J. Biol. Chem. 268:13344-13351 1993!). TFPI and variants have been shown to affecthemostasis in animal models of arterial reocclusion after thrombolysis(Haskel, E. J. et al., Circulation 84: 821-827 1991!), venous thrombosis(Holst, J. et al., Haemostasis 23 (suppl 1): 112-117 1993!), anddisseminated intravascular coagulation resulting from septic shock(Creasey, A. A. et al., J. Clin. Invest. 91: 2850-2860 1993!). However,TFPI may not have all of the properties desired for an anticoagulantagent for the treatment of thrombotic disease. The dependence of FXainhibition by TFPI prior to the inhibition of TF-FVIIa may haveundesirable effects. For instance, since FXa is produced by both theintrinsic and extrinsic pathways, inhibition of FXa may totally inhibitcoagulation and lead to undesirable side effects such as bleeding; aselective inhibitor of the extrinsic or intrinsic pathways may not leadto this problem. In addition heparin may affect the activity of TFPI.The carboxyl terminus of TFPI may be required for maximal activity(Wesselschmidt, R. et al., Blood 79:2004-2010 1992!; Nordfang, O. etal., Biochemistry 30: 10371-10376 1991!) Furthermore, TFPI is cleaved byhuman leukocyte elastase between the first two Kunitz domains whichresults in the loss of TF-FVIIa inhibitory activity (Higuchi, D. A. etal., Blood 79: 1712-1719 1992!).

Bovine pancreatic trypsin inhibitor (BPTI), also referred to asaprotinin, has recently been shown to competitively inhibit TF-FVIIaactivity, albeit with relatively weak affinity (Ki=30 μM) (Chabbat, J.et al., Thromb Res 71: 205-215 1993!). In addition, BPTI has recentlybeen shown to inhibit TF-induced coagulation; however, ca. 75 μM wasneeded to prolong the clotting time 1.4-fold in a PT assay (van denBesselaar, A. M. H. P. et al., Thromb Haemostas 69: 298-299 1993!).

Factor XIa

Factor XIa (FXIa) is a glycosylated serine protease produced in bloodfrom its zymogen, Factor XI (FXI). It is composed of a homodimer of twoidentical disulfide-linked proteins each having a molecular weight of80,000 Da (Kitchens, C. S., Semin. Thromb. Hemostas. 17: 55-72 1991!).In blood, most of the protein circulates bound to high molecular weightkininogen (HMWK). FXI is normally present at a concentration of ca. 4.5μg/ml. It can be activated by a number of serine proteases; FXIIa isthought to be a major activator, although thrombin has also recentlybeen implicated (Galiani, D. and Broze Jr., G. J., Science 253: 909-9121991!). In the presence of high molecular weight kininogen, FXIIa canactivate prekallikrein to kallikrein and FXI to FXIa; the kallikreinformed can activate more Factor XII to FXIIa. FXIa activates FIX toFIXa, which in the presence of Factor VIII leads to the formation of FXaand ultimately a fibrin clot (see FIG. 1).

The major physiological inhibitor of FXIa is thought to be the serpinα1-antitrypsin, also known as α1-proteinase inhibitor (Scott, C. F. etal., J. Clin. Invest. 69: 844-852 1982!). Serpins (serine proteaseinhibitors) such as α₁ -proteinase inhibitor have been wellcharacterized for their ability to inhibit various proteases because oftheir therapeutic potential to control proteolysis in thrombosis, shock,and inflammation (Schapira, M. et al., Trends Cardiovasc. Med.,4:146-151 1991!; Patston, P. A. et al., J. Biol. Chem. 265:10786-107911990!) and because spontaneous mutations to the P₁ residue (M358R; α₁-proteinase inhibitor-Pittsburgh) dramatically alter the proteaseinhibitor specificity (Scott, C. F. et al., J. Clin. Invest. 77:631-6341986!). However other serpins may inhibit FXIa; these include Clinhibitor, α2-antiplasmin, and antithrombin-III. α2-macroglobulin isanother inhibitor of FXIa. A low molecular weight inhibitor termed PIXIhas also been characterized from platelets (Cronlund, A. L. and Walsh,P. N., Biochemistry 31: 1685-1694 1992!).

Another inhibitor of FXIa is protease nexin-2, the secreted form of theAlzheimer's amyloid β-protein precursor, sometimes referred to asAβPP₇₅₁ and AβPP₇₇₀ for the different isoforms of this protein (VanNostrand, W. E. et al., J. Biol. Chem. 265: 9591-9594 1990!; Wagner, S.L. et al., Biochem. Biophys. Res. Commun. 186: 1138-1145 1992!; Smith,R. P. et al, Science 248: 1126-1128 1990!). This protein contains aKunitz domain which has been designated KPI (61 residues)(Wagner, S. L.et al., Biochem. Biophys. Res. Commun. 186: 1138-1145 1992!) or APPI (58residues) (Hynes, T. R. et al., Biochemistry 29: 10018-10022 1990!). TheKPI domain itself is also a potent inhibitor of FXIa (Wagner, S. L. etal., Biochem. Biophys. Res. Commun. 186: 1138-1145 1992!). Heparin hasbeen shown to potentiate the inhibition of FXIa by protease nexin-2, butnot by the KPI domain itself (Wagner, S. L. et al., Biochem. Biophys.Res. Commun. 186: 1138-1145 1992!). A variant of BPTI having arginine atposition 15 has been made semisynthetically and found to inhibit FXIawith relatively high affinity (Scott, C. F. et al., Blood 69: 1431-14361987!).

Kallikrein

Prekallikrein is a glycoprotein comprised of a single polypeptide chainwith a molecular weight of 80,000 Da and is present in normal plasma ata concentration of ca. 50 μg/ml (600 nM). In blood, 75 % ofprekallikrein circulates bound to HMWK. It is a serine protease zymogenwhich can be activated by FXIIa (see FIG. 1). Kallikrein consists of 2disulfide bonded chains of 43,000 and 33,000-36,000 Da. The light chainof kallikrein contains the enzymatic domain while the heavy chainappears to be required for surface dependent activation of coagulation.

Kallikrein cleaves HMWK to form bradykinin (a potent vasodilator andendothelial cell activator), can activate prourokinase and plasminogen(fibrinolytic), and feeds back for reciprocal activation of surfacebound FXII to FXIIa (see FIG. 1). In addition it can also stimulateneutrophils causing the release of elastase. Both Factor XIIa andkallikrein can lead to plasmin generation causing fibrinolysis.

The major physiological inhibitor of kallikrein is the serpin C1inhibitor, which inhibits irreversibly. In a purified system HMWK hasbeen shown to protect kallikrein from inhibition by C1 inhibitoralthough both proteins bind to kallikrein at different sites.α2-macroglobulin is another major inhibitor of kallikrein.Antithrombin-III can also inhibit kallikrein, but slowly even in thepresence of heparin. α2-antiplasmin and α1-antitrypsin are poorinhibitors of kallikrein. A mutant form of α₁ -proteinase inhibitor (α₁-proteinase inhibitor-Pittsburgh) that contains an Arg in the P₁position and an Ala in the P₂ position has been shown to be a morepotent inhibitor of Factor XIIf (FXIIf) and kallikrein compared to C1inhibitor, the most potent known natural inhibitor of these proteases(Schapira, M. et al., J. Clin. Invest 80:582-585 1987!; Patston, P. A.et al., J. Biol. Chem. 265:10786-10791 1990!). Rats treated with thismutant were partially protected from the hypotension resulting frominjection of FXIIf.

Basic pancreatic trypsin inhibitor (BPTI, aprotinin) reversibly inhibitsplasma kallikrein as well as plasmin and a number of other serineproteases; the P₁ residue of BPTI is a Lys. A variant of BPTI havingarginine at position 15 has been made semisynthetically and found toinhibit plasma kallikrein with a Ki of 15 nM, about 20-fold higheraffinity than BPTI (Scott, C. F. et al., Blood 69: 1431-1436 1987!).BPTI has been used to treat patients with acute pancreatitis (Fritz, H.and Wunderer, G., Arzneim.-Forsch. Drug Res. 33:479-494 1983!). The useof aprotinin and the possible involvement of the contact pathway (seebelow) has also been described for the reduction of bleeding frompostoperative surgery (Royston, D. Blood Coag. Fibrinol. 1:55-69 1990!)and in cardiopulmonary bypass surgery and for use in extracorporealcirculation models (Fuhrer, G. et al., Blood Coag. Fibrinol. 3:99-1041992!; Wachtfogel, Y. T. et al., J. Thorac. Cardiavasc. Surg. 106: 1-101993!). Similarly, soybean trypsin inhibitor has been shown to inhibitbradykinin formation and the initial hypotension induced by endotoxin inrats (Katori, M. et al., Br. J. Pharmacol. 98:1383-1391 1989!).

TF-FVIa in Disease (Thrombosis)

The formation of the TF-FVIIa complex is thought to be the key eventinitiating the coagulation cascade (Carson, S. D. and J. P. Brozna,Blood Coag Fibrinol 4: 281-292 1993; Davie, E. W. et al., Biochemistry30: 10363-10370 1991; Rapaport, S. I. and L. V. M. Rao, Arterioscler.Thromb. 12: 1111-1121 1992!). TF is found on the surface of theendothelium as well as monocytes and may become activated during theinflammatory response (Altieri, D. A., Blood 81: 569-579 1993!). Thus,inhibitors of the TF-FVIIa complex may be useful as anticoagulants andas antiinflammatory agents. A monoclonal antibody to TF has been shownto prevent mortality in a baboon model of septic shock (Taylor Jr., F.B. et al., Circ. Shock 33:127-134 1991!). A TF antibody has also shownthat TF may play a role in focal cerebral ischemia (Thomas, W. S. etal., Stroke 24: 847-854 1993!). In a rabbit model of thrombosis, amonoclonal antibody against rabbit TF inhibited thrombus formation incarotid arteries (Pawashe, A. B. et al., Circ. Res. 74: 56-63 1994!).

Contact Activation Pathways in Disease

Contact activation is a surface mediated pathway responsible in part forthe regulation of inflammation and thrombosis as mediated bycoagulation, kinin, fibrinolysis, complement, and other relevantpathways (see FIG. 1). The proteins involved in this pathway includeFXII (Hageman Factor), prekallikrein (Fletcher Factor), FXI, highmolecular weight kininogen (HMWK), and C1 inhibitor (DeLa Cadena, R. A.,et al. in Hemostasis and Thrombosis: Basic Principles and ClinicalPractice (Colman, R. W., Hirsh, J., Marder, V., & Salzman, E. W., eds.)pp. 219-240, J. B. Lippincott Co., Philadelphia 1994!; Wachtfogel, Y. T.et al., Thromb. Res. 72: 1-21 1993!). The involvement of this plasmaprotease system has been suggested to play a significant role in avariety of clinical manifestations including septic shock, adultrespiratory distress syndrome (ARDS), disseminated intravascularcoagulation (DIC), cardiopulmonary bypass surgery, bleeding frompostoperative surgery, and various other disease states (Colman, R. W.,N. Engl. J. Med 320:1207-1209 1989!; Bone, R. C., Arch. Intern. Med.152:1381-1389 1992!).

Septic shock

Septic shock is the most common cause of death of humans in intensivecare units in the United States (Parillo, J. E. et al., Ann. Tnt. Med.113:227-242 1990!; Schmeichel C. J. and McCormick D., BioTechnol.10:264-267 1992!). It is usually initiated by a local nidus of infectionthat invades the blood stream. Incidences of sepsis and shock can arisefrom infections with either gram negative, gram positive bacterial orfungal microorganisms. All these organisms seem to induce a commonpattern of cardiovascular dysfunction. In recent years aggressive fluidinfusion therapy has been accepted as a primary means of treatment forseptic shock. Adequate repletion of fluid is associated with an elevatedcardiac output and low vascular resistance. Despite treatment, septicshock results in a severe decrease in systemic vascular resistance andgeneralized blood flow maldistribution. Aggressive therapy reversesshock and death in about 50% of the cases. Unresponsive hypotensionresulting from a very low vascular resistance cannot be corrected byfluid infusion. Among those subjects that die from septic shock,approximately 75% die from persistent hypotension and the remainder dueto multiple organ system failure (see FIG. 1).

The increase in cardiac output and vasodilation in septic shock isattributed to the action of inflammatory mediators. While the actualevents leading to septic shock, DIC and hypotension have not beenestablished, the known interactions among various components of the manyphysiological systems suggest that activation of the contact pathway maylead to a state of septic shock, multiorgan failure, and death (Bone, R.C., Arch. Intern. Med. 152:1381-1389 1992!) as illustrated in FIG. 1.The contact system of intrinsic coagulation and the complement systemare excessively activated in sepsis and septic shock, especially incases of fatal septic shock. The contact system can participate in thegeneration of many vasoactive mediators such as bradykinin, FXIIa, FXIIfand C5a, which are thought to play a role in the pathogenesis of fatalshock. Bradykinin, FXIIa, and FXIIf are potent inducers of hypotensionwhile C5a is an inducer of vasodilation and vasopermeability. The levelsof FXII, prekallikrein, and high molecular weight kininogen aredecreased significantly during non-fatal shock, but are most severelydepressed during fatal septic shock to approximately 30%, 57% and 27% ofnormal values respectively. These changes are noted regardless ofwhether the septic state is caused by gram positive or gram negativebacteria. The contact activation pathway is also involved in both fibrindeposition and lysis, as well as triggering neutrophil activation,activation of complement and modulation of blood pressure.

Decreased levels of prekallikrein are observed in hepatic disease, DIC,chronic renal failure and nephritic syndrome. In septic shock,components of the kallikrein-kinin system are depleted suggestingactivation of this system. This is not the case in cardiogenic shocksuggesting that the kallikrein-kinin system is a key player in septicshock (Martinez-Brotons F. et al., Thromb. Haemostas. 58:709-713 1987!)

ARDS

ARDS is a complex pulmonary disorder affecting 150,000 people in theU.S. yearly with a 50% mortality rate. Leukocytes, platelets and theproteolytic pathways of coagulation and complement mediate ARDS. ARDSinvolves activation of the contact activation pathway and depletion ofC1 inhibitor. Sepsis induced ARDS results in more severe DIC andfibrinolysis, more fibrin degradation products and reduced ATIII levelscompared to trauma induced ARDS (Carvalho, A. C. et al., J. Lab. Clin.Med. 112:270-277 1988!).

Disseminated Intravascular Coagulation

Disseminated intravascular coagulation (DIC) is a disorder that occursin response to tissue injury and invading microorganisms characterizedby widespread deposition of fibrin and depleted levels of fibrinogen(Muller-Berghaus, G. Semin. Thromb. Hemostasis 15:58-87 1989!). Thereare prolonged prothrombin and activated partial thromboplastin times.DIC has been observed in the clinical settings of a wide variety ofdiseases (Fruchtman, S. M. and Rand, J. H. in Thrombosis inCardiovascular Disorders (Fuster, V. and Verstraete M., eds.) pp.501-513 W. B. Saunders, Philadelphia 1992!).

Hypotension, DIC, and neutrophil activation are all triggered by theinteraction of Factor XIIa, plasma kininogens and kallikrein. Deficiencyof any of these 3 proteins does not give rise to hemostatic disordersdue to redundancy in the system due to platelets, other coagulationfactors, and endothelial cells.

A large number of therapeutic approaches to septic shock and relateddisorders have been identified including various cytokine antagonists,Mabs (to endotoxin, tissue factor, tumor necrosis factor (TNF),neutrophils, etc.), kinin antagonists, bactericidal permeabilityincreasing protein, PAF antagonists, C1 inhibitor, DEGR-FXa, activatedprotein C, and many other approaches. It is possible, due to thecomplicated nature of the disease, that an approach that involvesmultiple agents or agents that effect multiple pathways may besuccessful in the treatment of septic shock (Schmeichel C. J. andMcCormick D., BioTechnol. 10:264-267 1992!).

Kunitz Domain Inhibitors of Serine Proteases

The Kunitz-type protease inhibitor domains found in TFPI are found amongother mammalian proteins including BPTI, Alzheimer's amyloid β-proteinprecursor, and inter-α-trypsin inhibitor (Creighton, T. E. and I. G.Charles, Cold Spring Harbor Symp. Quant. Biol. 52: 511-519 1987!;Salvesen, G. and Pizzo, S., in Hemostasis and Thrombosis: BasicPrinciples and Clinical Practice (Colman, R. W., Hirsh, J., Marder, V.,& Salzman, E. W., eds.) pp. 241-258, J. B. Lippincott Co., Philadelphia1994!) (FIG. 2). Kunitz-type protease inhibitors have also been preparedfrom the α-3 chain of human type VI collagen (see WO 93/14119). Theyhave also been identified in many snake venoms. Recently, Kunitzinhibitors of TF-FVIIa have been prepared from BPTI using phage displaytechnology (De Maeyer et al., Thrombosis and Haemostasis Abstracts,XIV^(th) Congress of the International Society on Thrombosis andHaemostasis, p 888 Ab. No. 1245 1993!). These authors report a mutantBPTI (Thr11Asp, Lys15Arg, Arg17Leu, Ile18His, Ile19Leu, Va134Tyr,Arg39Leu and Lys46Glu) having a K_(i) for TF-FVIIa of 0.5 nM.

Kunitz domains are generally stable proteins containing about 60residues and six specifically spaced cysteines that are present indisulfide bonds. They are known to be slow, tight-binding, reversibleinhibitors of serine proteases that bind to the active site and inhibitaccording to the standard mechanism. Subsequent cleavage between the P₁and P₁ ' residues occurs very slowly if at all (Bode, W. and Huber, R.,Eur. J. Biochem. 204: 433-451 1992!; Laskowski, M., Jr. and Kato, I.,Annu. Rev. Biochem. 49: 593-626 1980!). There are many interactionsbetween the serine protease subsites and the side chains in the primarybinding loop of Kunitz domains (P₅ -P₄ ') (Bode, W. and Huber, R., Eur.J. Biochem. 204: 433-451 1992!; Laskowski, M., Jr. and Kato, I., Annu.Rev. Biochem. 49: 593-626 1980!); however, the interactions of the P₁residue with the specificity pocket are energetically most important andtherefore represent the primary specificity determinants (see FIG. 3).Substrates and inhibitors of TF-FVIIa and other trypsin-like proteasessuch as FXIa and kallikrein have either Arg or Lys at the P₁ residue.Therefore, at position 15 (P₁), either Arg or Lys is generallypreferred. However methionine is sometimes found at the P₁ position andmay also be preferable for good inhibition of serine proteases (McCrath,M. E. et al., J. Biol. Chem. 266:6620-6625 1991!). The introduction ofresidues such as Val, Leu, or Ile at the P₁ position of Kunitz domainsleads to potent inhibitors of human leukocyte elastase (HLE) andconcomitant loss of the wild type inhibitory activity (Beckmann, J. etal., Eur. J. Biochem. 176: 675-682 1988!; Sinha, S. et al., J. Biol.Chem. 266: 21011-21013 1991!). Residues other than naturally occurringamino acids have also been substituted into Kunitz domains and otherrelated protease inhibitor domains by chemical synthesis (Beckmann, J.et al., Eur. J. Biochem. 176: 675-682 1988!; Bigler, T. L. et al., Prot.Sci. 2: 786-799 1993!).

The crystal structures of Kunitz domains reveal key residues likely tomake contact with the serine protease domain of FVIIa and other serineproteases (Hynes, T. R. et al., Biochemistry 29: 10018-10022 1990; Bode,W. and Huber, R., Eur. J. Biochem. 204: 433-451 1992!; Kossiakoff, A. A.et al., Biochem Soc Trans 21: 614-618 1993!). Although the amino acid atthe P₁ position generally dominates the affinity of inhibitors for theserine protease active site (Scott, C. F. et al., Blood 69: 1431-14361987; Laskowski, M., Jr. and Kato, I., Annu. Rev. Biochem. 49: 593-6261980!; Beckmann, J. et al., Eur. J. Biochem. 176: 675-682 1988!; Sinha,S. et al., J. Biol. Chem. 266: 21011-21013 1991!), residues outside thisregion are also known to play a role in binding affinity and specificitytowards serine proteases (Kossiakoff, A. A. et al., Biochem. Soc. Trans.21: 614-618 1993!; Roberts, B. L. et al., Proc Natl Acad Sci USA 89:2429-2433 1992!). Some of the contact residues in the binding loop(positions 11, 15, 17, and 19) are relatively variable among Kunitzdomains (Creighton, T. E. and I. G. Charles, Cold Spring Harbor Symp.Quant. Biol. 52: 511-519 1987!). Position 13 is normally a Pro; however,other residues are sometimes found here. Position 12 is almost always aGly. In addition to recruiting any side chain interactions, substitutionof other residues for Pro and vice versa might also lead toconformational changes in the main chain which could affect binding. Thecysteine residues at positions 14 and 38 that form a disulfide bond arealways found in Kunitz domains; however other residues such as Ala, Gly,Ser, or Thr may substitute for the cysteines (Marks, C. B. et al.,Science, 235: 1370-1373 1987!).

In APPI and other Kunitz domains, residues 13 and 39 as well as residues17 and 34 are in close proximity (FIG. 3) (Hynes, T. R. et al.,Biochemistry 29: 10018-10022 1990!). Therefore, the potentialinteractions of residues 34 and 39 with the primary binding loop of APPIwere investigated to address whether these positions would affectbinding. Residues at positions 16 and 18 are generally more invariantamong Kunitz domains (Creighton, T. E. and I. G. Charles, Cold SpringHarbor Symp. Quant. Biol. 52: 511-519 1987!); however, differentresidues at these positions may also alter binding. Therefore, residuesat positions 11 through 19, 34, 38, and 39 may all affect the bindingaffinity and specificity towards serine proteases (FIG. 3). However,other residues are important as well. For instance, APPI and BPTI have amethionine at position 52, although other Kunitz domains have a varietyof residues at this position (FIG. 2). Methionine at this position canbe replaced by different residues which may be beneficial with respectto producing the protein. For example, methionine is susceptible tooxidation to form methionine sulfoxide, which can complicatepurification. Also protein can be made recombinantly as a fusionprotein, followed by cleavage with CNBr, which cleaves at methionineresidues (Auerswald, E. A. et al., Biol. Chem. Hoppe-Seyler 369: 27-351988!). Therefore, it is necessary to remove other methionine residuesin the protein of interest to produce intact product. Substitutions atposition 52 are not expected to have major effects on inhibitoryactivity since it is so far away from the primary binding loop of theKunitz domain (FIG. 3).

The 61 residue Kunitz protease inhibitor domain of the Alzheimer'samyloid β-protein precursor (KPI), binds to the active site of mammalianserine proteases trypsin, chymotrypsin and Factor XIa with high affinity(Wagner, S. L. et al., Biochem. Biophys. Res. Commun. 186: 1138-11451992!). Similar results were found with a fusion protein containing thisdomain (Sinha, S. et al., J. Biol. Chem. 266: 21011-21013 1991!). TheKPI domain has also been shown to inhibit FIXa activity, although muchless potently than protease nexin-2, from which it was derived (SchmaierA. H. et al., J. Clin. Invest. 92: 2540-2545 1993!). The KPI domain at100 μM independently inhibited the coagulant activity of both Factor Xaand VIIa in plasma more than twofold over control. However, thisinhibition was at least two orders of magnitude weaker than theinhibition of Factor XIa by the KPI domain, which at ˜0.5 μM resulted ina twofold prolongation of the Factor XIa coagulant assay. We chose APPIas a scaffold since (a) it has been readily expressed in bacteria suchas E. coli (Castro, M. et al., FEBS Lett. 267: 207-212 1990!) and yeastsuch as P. pastoris, (b) an x-ray crystal structure of the protein isknown (Hynes, T. R. et al., Biochemistry 29: 10018-10022 1990!), and (c)it is derived from a human sequence, which would minimize theimmunogenicity for any therapeutically useful variants. Other Kunitzdomains from human and other mammalian sources may be used similarly.

Accordingly, it is an object of this invention to provide potent serineprotease inhibitors that reversibly inhibit proteases of thecoagulation, contact activation, fibrinolysis, inflammation, complementactivation, and hypotensive pathways for the treatment of diseases thatare affected by these pathways. It is further an object of thisinvention to provide potent inhibitors capable of inhibiting FactorVIIa, Factor XIa, kallikrein, and plasmin. Additionally, it is an objectto provide synthetic methods for producing these inhibitors fortherapeutic intervention. These and other objects will be apparent fromconsideration of this application as a whole.

SUMMARY OF THE INVENTION

By means of the present invention the objectives described above havebeen realized, and there is accordingly provided herein a composition ofmatter capable of inhibiting a serine protease selected from FactorVIIa, Factor XIa, plasma kallikrein, and plasmin, comprising a purifiedpolypeptide having an amino acid sequence represented by StructuralFormula I:

R₁ -Xaa₁₁ -Xaa₁₂ -Xaa₁₃ -Xaa₁₄ -Xaa₁₅ -Xaa₁₆ -Xaa₁₇ -Xaa₁₈ -Xaa₁₉ -R₂-Xa a ₃₄ -R₃ -Xaa₃₈ -Xaa₃₉ -R₄

where

R₁ represents a peptide comprising from 5 to 250 naturally occurringamino acid residues wherein at least one residue is Cys;

R₂ represents a peptide having 14 amino acid residues wherein at leastone residue is Cys;

R₃ represents a tripeptide;

R₄ represents a peptide comprising from 12 to 250 amino acid residueswherein at least one residue is Cys;

Xaa₁₁ is a naturally occurring amino acid residue selected from thegroup Pro, Arg, Ala, Glu, Gly, and Thr;

Xaa₁₂ represents Gly;

Xaa₁₃ is a naturally occurring amino acid residue selected from thegroup Pro, Leu, Trp, Val, Gly, Phe, His, Tyr, Ala, Ile, Glu, and Gln;

Xaa₁₄ is a naturally occurring amino acid residue selected from Cys,Ala, Ser, Thr, and Gly;

Xaa₁₅ is a naturally occurring amino acid residue selected from Met,Arg, and Lys;

Xaa₁₆ is a naturally occurring amino acid residue selected from Gly andAla;

Xaa₁₇ is a naturally occurring amino acid residue selected from thegroup Met, Leu, Ile, Arg, Tyr, and Ser;

Xaa₁₈ is a naturally occurring amino acid residue selected from thegroup Ile, His, Leu, Met, Tyr, and Phe;

Xaa₁₉ is a naturally occurring amino acid residue selected from thegroup Leu, Arg, Ala, Lys, and Ile;

Xaa₃₄ is a naturally occurring amino acid residue selected from thegroup Phe, Ile, Ser, Leu, Tyr, Trp, and Val;

Xaa₃₈ is a naturally occurring amino acid residue selected from Cys,Ala, Ser, Thr, and Gly; and

Xaa₃₉ is a naturally occurring amino acid residue selected from thegroup Tyr, Gly, Trp, His, and Phe; provided

R₁ is not Xaa¹ -Asp-Ile-Cys-Lys-Leu-Pro-Lys-Asp (SEQ ID NO: 1), whereXaa¹ is His or 1-5 amino acid residues; and

Xaa₁₁ through Xaa₁₉ are not

Pro-Gly-Phe-Ala-Lys-Ala-Ile-Ile-Arg (SEQ ID NO: 2);

Thr-Gly-Leu-Cys-Lys-Ala-Tyr-Ile-Arg (SEQ ID NO: 3);

Thr-Gly-Leu-Cys-Lys-Ala-Arg-Ile-Arg (SEQ ID NO: 4); and

Ala-Gly-Ala-Ala-Lys-Ala-Leu-Leu-Ala (SEQ ID NO: 5).

A preferred polypeptide represented by Formula I has an apparentdissociation constant (K_(i) ^(*)) with respect to tissue factor-FactorVIIa of less than about 100 nM, more preferably less than 10 nM and mostpreferably 3 nM or lower. Optionally, the preferred polypeptide also hasan apparent dissociation constant (K_(i) ^(*)) with respect to bothFactor XIa and kallikrein of less than about 10 nM and most preferably 2nM or lower. Polypeptides of Formula I that are potent inhibitors of allthree of; TF-FVIIa, FXIa, and kallikrein preferably have Xaa₁₈ -Xaa₁₉ asIle-Leu. Optionally, the preferred polypeptide specifically inhibitsTF-FVIIa and has an apparent dissociation constant (K_(i) ^(*)) withrespect to both Factor XIa and kallikrein of greater than about 50 nMand most preferably greater than about 80 nM. Polypeptides of Formula Ithat are specific potent inhibitors of TF-FVIIa preferably have Xaa₁₈-Xaa₁₉ as Met-Lys/Arg.

The preferred polypeptide represented by Structural Formula I comprisesabout 58 amino acid residues in which R₁ is a 10 residue peptide, R₂ isa 14 residue peptide, R₃ is a tripeptide, and R₄ is a 19 residuepeptide, and where residue 5, 14, 30, 38, 51 and 55 are Cys. Alsopreferably, residues 12 and 37 are G, residues 33 and 45 are Phe,residue 35 is Tyr and residue 43 is Asn.

Exemplary preferred polypeptides of Structural Formula I are as follows:

R₁ is selected from the group

Val-Arg-Glu-Val-Cys-Ser-Glu-Gln-Ala-Glu (SEQ ID NO: 6);

Met-His-Ser-Phe-Cys-Ala-Phe-Lys-Ala-Asp (SEQ ID NO: 7);

Lys-Pro-Asp-Phe-Cys-Phe-Leu-Glu-Glu-Asp (SEQ ID NO: 8);

Gly-Pro-Ser-Trp-Cys-Leu-Thr-Pro-Ala-Asp (SEQ ID NO: 9);

Lys-Glu-Asp-Ser-Cys-Gln-Leu-Gly-Tyr-Ser (SEQ ID NO: 10);

Thr-Val-Ala-Ala-Cys-Asn-Leu-Pro-Ile-Val (SEQ ID NO: 11);

Leu-Pro-Asn-Val-Cys-Ala-Phe-Pro-Met-Glu (SEQ ID NO: 12); and

Arg-Pro-Asp-Phe-Cys-Leu-Glu-Pro-Pro-Tyr (SEQ ID NO: 13);

R₂ is selected from the group

Arg-Trp-Tyr-Phe-Asp-Val-Thr-Glu-Gly-Lys-Cys-Ala-Pro-Phe (SEQ ID NO: 14);

Arg-Phe-Phe-Phe-Asn-Ile-Phe-Thr-Arg-Gln-Cys-Glu-Glu-Phe (SEQ ID NO: 15);

Arg-Tyr-Phe-Tyr-Asn-Asn-Gln-Thr-Lys-Gln-Cys-Glu-Arg-Phe (SEQ ID NO: 16);

Arg-Phe-Tyr-Tyr-Asn-Ser-Val-Ile-Gly-Lys-Cys-Arg-Pro-Phe (SEQ ID NO: 17);

Arg-Tyr-Phe-Tyr-Asn-Gly-Thr-Ser-Met-Ala-Cys-Glu-Thr-Phe (SEQ ID NO: 18);

Leu-Trp-Ala-Phe-Asp-Ala-Val-Lys-Gly-Lys-Cys-Val-Leu-Phe (SEQ ID NO: 19);

Lys-Trp-Tyr-Tyr-Asp-Pro-Asn-Thr-Lys-Ser-Cys-Ala-Arg-Phe (SEQ ID NO: 20;

Arg-Trp-Phe-Phe-Asn-Phe-Glu-Thr-Gly-Glu-Cys-Glu-Leu-Phe (SEQ ID NO: 21);and

Arg-Tyr-Phe-Tyr-Asn-Ala-Lys-Ala-Gly-Leu-Cys-Gln-Thr-Phe (SEQ ID NO: 22);

R₃ is selected from the group

Tyr-Gly-Gly; and

Tyr-Ser-Gly;

R₄ is selected from the group

Gly-Asn-Arg-Asn-Asn-Phe-Asp-Thr-Glu-Glu-Tyr-Cys-Ala-Ala-Val-Cys-Gly-Ser-Ala(SEQ ID NO: 23);

Gly-Asn-Arg-Asn-Asn-Phe-Asp-Thr-Glu-Glu-Tyr-Cys-Met-Ala-Val-Cys-Gly-Ser-Ala(SEQ ID NO: 24);

Gly-Asn-Gln-Asn-Arg-Phe-Glu-Ser-Leu-Glu-Glu-Cys-Lys-Lys-Met-Cys-Thr-Arg-Asp(SEQ ID NO: 25);

Gly-Asn-Met-Asn-Asn-Phe-Glu-Thr-Leu-Glu-Glu-Cys-Lys-Asn-Ile-Cys-Glu-Asp-Gly(SEQ ID NO: 26);

Gly-Asn-Glu-Asn-Asn-Phe-Thr-Ser-Lys-Gln-Glu-Cys-Leu-Arg-Ala-Cys-Lys-Lys-Gly(SEQ ID NO: 27);

Gly-Asn-Gly-Asn-Asn-Phe-Val-Thr-Glu-Lys-Glu-Cys-Leu-Gln-Thr-Cys-Arg-Thr-Val(SEQ ID NO: 28);

Gly-Asn-Gly-Asn-Lys-Phe-Tyr-Ser-Glu-Lys-Glu-Cys-Arg-Glu-Tyr-Cys-Gly-Val-Pro(SEQ ID NO: 29);

Gly-Asn-Glu-Asn-Lys-Phe-Gly-Ser-Gln-Lys-Glu-Cys-Glu-Lys-Val-Cys-Ala-Pro-Val(SEQ ID NO: 30);

Gly-Asn-Ser-Asn-Asn-Phe-Leu-Arg-Lys-Glu-Lys-Cys-Glu-Lys-Phe-Cys-Lys-Phe-Thr(SEQ ID NO: 31); and

Ala-Lys-Arg-Asn-Asn-Phe-Lys-Ser-Ala-Glu-Asp-Cys-Met-Arg-Thr-Cys-Gly-Gly-Ala(SEQ ID NO: 32);

Xaa₁₁ is Pro;

Xaa₁₂ is Gly;

Xaa₁₃ is selected from the group Pro, Val, Leu, and Trp;

Xaa₁₄ is Cys;

Xaa₁₅ is Arg or Lys;

Xaa₁₆ is Ala;

Xaa₁₇ is Met or Leu;

Xaa₁₈ is Met or Ile;

Xaa₁₉ is Leu, Lys or Arg;

Xaa₃₄ is Phe, Val, Ile, or Tyr;

Xaa₃₈ is Cys; and

Xaa₃₉ is Tyr, Gly, or His.

In a further embodiment polypeptides of the sequence represented by:

R₁ -Xaa₁₁ -Xaa₁₂ -Xaa₁₃ -Xaa₁₄ -Xaa₁₅ -Xaa₁₆ -Xaa₁₇ -Xaa₁₈ -Xaa₁₉ -R₂Xaa ₃₄ -R₃ -Xaa₃₈ -Xaa₃₉ -R₄

where in each case R₁ has the sequence:

Val-Arg-Glu-Val-Cys-Ser-Glu-Gln-Ala-Glu (SEQ ID NO: 6)

R₂ has the sequence:

Arg-Trp-Tyr-Phe-Asp-Val-Thr-Glu-Gly-Lys-Cys-Ala-Pro-Phe (SEQ ID NO: 14)

R₃ has the sequence:

Tyr-Gly-Gly; and

R₄ has the sequence:

Gly-Asn-Arg-Asn-Asn-Phe-Asp-Thr-Glu-Glu-Tyr-Cys-Ala-Ala-Val-Cys-Gly-Ser-Ala(SEQ ID NO: 23) or

Gly-Asn-Arg-Asn-Asn-Phe-Asp-Thr-Glu-Glu-Tyr-Cys-Met-Ala-Val-Cys-Gly-Ser-Ala(SEQ ID NO: 24)

are especially preferred. Therefore, polypeptides having the followingdesignations and structural formulas are preferred:

I-18 R₁ -Pro-Gly-Val-Cys-Arg-Ala-Leu-Ile-Leu-R₂ - Phe-R₃ -Cys-Gly-R₄(SEQ ID NO: 43)

I-49 R₁ -Pro-Gly-Trp-Cys-Arg-Ala-Leu-Ile-Lue-R₂ -Phe-R₃ -Cys-Gly-R₄ (SEQID NO: 44)

I-14 R₁ -Pro-Gly-Phe-Cys-Arg-Ala-Leu-Ile-Leu-R₂ -Phe-R₃ -Cys-Gly-R₄ (SEQID NO: 45)

I-16 R₁ -Gly-Gly-Trp-Cys-Arg-Ala-Leu-Ile-Leu-R₂ - Phe-R₃ -Cys-Gly-R₄(SEQ ID NO: 46)

where R₄ is the sequence identified by SEQ ID NO: 24, and

II-4 R₁ -Pro-Gly-Pro-Cys-Arg-Ala-Met-Ile-Ser-R₂ - Phe-R₃ -Cys-Tyr-R₄(SEQ ID NO: 47)

II-3 R₁ -Pro-Gly-Trp-Cys-Arg-Ala-Met-Ile-Ser-R₂ -Ile-R₃ -Cys-Gly-R₄ (SEQID NO: 48)

II-6 R₁ -Pro-Gly-Pro-Cys-Lys-Ala-Met-Ile-Ser-R₂ -Ile-R₃ -Cys-Trp-R₄ (SEQID NO: 49)

III-27 R₁ -Thr-Gly-Pro-Cys-Arg-Ala-Leu-Ile-Ser-R₂ -Trp-R₃ -Cys-Gly-R₄(SEQ ID NO: 50)

III-30 R₁ -Thr-Gly-Pro-Cys-Arg-Ala-Leu-Ile-Ser-R₂ -Tyr-R₃ -Cys-Gly-R₄(SEQ ID NO: 51)

TF7I-VY R₁ -Pro-Gly-Val-Cys-Arg-Ala-Leu-Ile-Leu-R₂ -Phe-R₃ -Cys-Tyr-R₄(SEQ ID NO: 52)

TF7I-LY R₁ -Pro-Gly-Leu-Cys-Arg-Ala-Leu-Ile-Leu-R₂ -Phe-R₃ -Cys-Tyr-R₄(SEQ ID NO: 53)

TF7I-WY R₁ -Pro-Gly-Trp-Cys-Arg-Ala-Leu-Ile-Leu-R₂ -Phe-R₃ -Cys-Tyr-R₄(SEQ ID NO: 54)

TF7I-PG R₁ -Pro-Gly-Pro-Cys-Arg-Ala-Leu-Ile-Leu-R₂ -Phe-R₃ -Cys-Gly-R₄(SEQ ID NO: 55)

IV-47C R₁ -Pro-Gly-Pro-Cys-Arg-Ala-Met-Met-Lys-R₂ -Ile-R₃ -Cys-His-R₄(SEQ ID NO: 56)

IV-54C R₁ -Pro-Gly-Pro-Cys-Arg-Ala-Leu-Met-Lys-R₂ -Val-R₃ -Cys-Tyr-R₄(SEQ ID NO: 57)

IV-31B R₁ -Pro-Gly-Pro-Cys-Arg-Ala-Leu-Met-Lys-R₂ -Val-R₃ -Cys-Phe-R₄(SEQ ID NO: 58)

IV-49C R₁ -Pro-Gly-Pro-Cys-Arg-Ala-Met-Met-Lys-R₂ -Ile-R₃ -Cys-Tyr-R₄(SEQ ID NO: 59)

IV-50C R₁ -Pro-Gly-Pro-Cys-Arg-Ala-Met-Tyr-Lys-R₂ -Ile-R₃ -Cys-Tyr-R₄(SEQ ID NO: 60)

IV-57C R₁ -Pro-Gly-Val-Cys-Arg-Ala-Met-Met-Lys-R₂ -Ile-R₃ -Cys-Gly-R₄(SEQ ID NO: 61)

IV-51C R₁ -Pro-Gly-Pro-Cys-Lys-Ala-Leu-Met-Arg-R₂ -Tyr-R₃ -Cys-Tyr-R₄(SEQ ID NO: 62)

IV-35B R₁ -Pro-Gly-Pro-Cys-Lys-Ala-Ile-Met-Lys-R₂ -Ile-R₃ -Cys-His-R₄(SEQ ID NO: 63)

IV-58C R₁ -Pro-Gly-Pro-Cys-Lys-Ala-Leu-Met-Lys-R₂ -Tyr-R₃ -Cys-His-R₄(SEQ ID NO: 64)

IV-48C R₁ -Pro-Gly-Pro-Cys-Lys-Ala-Leu-Met-Lys-R₂ -Trp-R,-Cys-Trp-R₄(SEQ ID NO: 65)

IV-46C R₁ -Pro-Gly-Pro-Cys-Lys-Ala-Met-Ile-Lys-R₂ -Leu-R₃ -Cys-Tyr-R₄(SEQ ID NO: 66)

IV-55C R₁ -Pro-Gly-Pro-Cys-Lys-Ala-Leu-Met-Lys-R₂ -Phe-R₃ -Cys-Tyr-R₄(SEQ ID NO: 67)

IV-32B R₁ -Pro-Gly-Pro-Cys-Lys-Ala-Leu-Met-Lys-R₂ -Tyr-R₃ -cys-Tyr-R₄(SEQ ID NO: 68)

IV-36B R₁ -Pro-Gly-Pro-Cys-Lys-Ala-Leu-Met-Lys-R₂ -Val-R₃ -Cys-Tyr-R₄(SEQ ID NO: 69)

IV-40B R₁ -Pro-Gly-Ala-Cys-Lys-Ala-Met-Tyr-Lys-R₂ -Ile-R₃ -Cys-Gly-R₄(SEQ ID NO: 70)

53b R₁ -Pro-Gly-Pro-Gly-Arg-Ala-Leu-Ile-Leu-R₂ -Phe-R₃ -Ala-Tyr-R₄ (SEQID NO: 71)

and TF71-IC R₁ -Pro-Gly-Pro-Cys-Arg-Ala-Leu-Ile-Leu-R₂ -Phe-R₃-Cys-Tyr-R₄ (SEQ ID NO: 72)

where R₄ is the sequence identified by SEQ ID NO: 23. However, as noted,according to the invention, R₁ can be any peptide having from 5 to 250amino acids residues wherein at least one residue is a Cys. In specificembodiments R₁ is selected from the group consisting of (SEQ ID NO: 6),(SEQ ID NO: 7), (SEQ ID NO: 8), (SEQ ID NO: 9), (SEQ ID NO: 10), (SEQ IDNO: 11), (SEQ ID NO: 12), or (SEQ ID NO: 13). R₂ can be any peptidehaving 14 amino acids wherein at least one residue is a Cys. In specificembodiments, R₂ is selected from the group consisting of (SEQ ID NO:14), (SEQ ID NO: 15), (SEQ ID NO: 16), (SEQ ID NO: 17), (SEQ ID NO: 18),(SEQ ID NO: 19), (SEQ ID NO: 20), (SEQ ID NO: 21), (SEQ ID NO: 22). R₃is a tripeptide, especially a tripeptide selected from the groupconsisting of Tyr-Gly-Gly, or Tyr-Ser-Gly. R₄ can be any peptide having12 to 250 amino acids wherein at least one residue is a Cys. In specificembodiments R₄ is selected from the group consisting of (SEQ ID NO: 23),(SEQ ID NO: 24), (SEQ ID NO: 25), (SEQ ID NO: 26), (SEQ ID NO: 27), (SEQID NO: 28), (SEQ ID NO: 29), (SEQ ID NO: 30), (SEQ ID NO: 31), (SEQ IDNO: 32).

The present invention also contemplates polypeptides where, in additionto the changes noted above, the residues corresponding to residue 20, 44and 46 of APPI are modified. According to this aspect of the presentinvention, amino acid substitutions that promote the favorableinteraction between the polypeptide and the particular serine proteaseof the coagulation cascade are contemplated. According to this aspect ofthe present invention residue 20, which is equivalent to the first aminoacid residue of R₂, is modified. Preferably, according to this aspect ofthe invention the amino acid at position 20 of APPI is modified to Ala,Val, Ser, Thr, Asn, Gln, Asp, Glu, Leu, or Ile. Therefore, variantswherein the first amino acid of R₂ is represented by Xaa₂₀, where Xaa₂₀is selected from the group consisting of Ala, Val, Ser, Thr, Asn, Gln,Asp, Glu, Leu, and Ile are preferred.

According to this aspect of the present invention residues 44 and 46 ofAPPI which are equivalent to the fifth and the seventh residues of R₄are modified to promote the favorable interaction between thepolypeptide and the particular serine protease of the coagulationcascade such as the tissue factor-factor VIIa complex. Therefore,polypeptides where positions 44 and 46, represented by Xaa₄₄ and Xaa₄₆,respectively, are modified to promote the favorable interaction betweenthe polypeptide and tissue factor-factor VIIa complex are preferred.Exemplary residues at Xaa₄₆ include Asp or Glu. Other substitutions willbe apparent to one of skill in the art based on the teachings of theinstant application. The resulting polypeptide has an apparent K_(i)^(*) with respect to Tissue Factor-Factor VIIa of less that about 100nM, more preferable less that 10 nM and most preferably 3 nM or lower.Optionally, the polypeptide also has an apparent K_(i) ^(*) with respectto both Factor XIa and kallikrein of less than about 10 nM and mostpreferably 2 nM or lower.

In a further embodiment, the present invention encompasses a compositionof matter comprising isolated nucleic acid, preferably DNA, encoding theprotein component of a composition of matter comprising a polypeptiderepresented by Structural Formula I. The invention further comprises anexpression control sequence operably linked to the DNA molecule, anexpression vector, preferably a plasmid, comprising the DNA molecule,where the control sequence is recognized by a host cell transformed withthe vector, and a host cell transformed with the vector.

Preferred expression vectors of the present invention may be selectedfrom; pBR322, phGH1, pBO475, pRIT5, pRIT2T, pKK233-2, pDR540, andpPL-lambda, with the most preferred vector being pSAlz1.

Preferred host cells containing the expression vector of the presentinvention may be selected from E. coli K12 strain 294 (ATCC No. 31446),E. coli strain JM101, E. coli B, E. coli X1776 (ATCC No. 31537), E. colic600, E. coli W3110 (F-, gamma-, prototrophic, ATCC No. 27325), Bacillussubtilis, Salmonella typhimurium, Serratia marcesans, and Pseudomonasspecies, with the most preferred host cell being E. coli W3110 (ATCC No.27325), or a derivative thereof such as the protease deficient strain27C7.

The composition of the present invention may be made by a process whichincludes the steps of isolating or synthesizing nucleic acid sequencesencoding any of the amino acid sequences described above, ligating thenucleic acid sequence into a suitable expression vector capable ofexpressing the nucleic acid sequence in a suitable host, transformingthe host with the expression vector into which the nucleic acid sequencehas been ligated, culturing the host under conditions suitable forexpression of the nucleic acid sequence, whereby the protein encoded bythe selected nucleic acid sequence is expressed by the host. In thisprocess, the ligating step may further contemplate ligating the nucleicacid into a suitable expression vector such that the nucleic acid isoperably linked to a suitable secretory signal, whereby the amino acidsequence is secreted by the host. The secretory signal may be selectedfrom the group consisting of the leader sequence of stII, lamB, herpesgD, lpp, alkaline phosphatase, invertase, and alpha factor and ispreferably stII.

The present invention further extends to therapeutic applications forthe compositions described herein. Thus the invention includes apharmaceutical composition comprising a pharmaceutically acceptableexcipient and a purified amino acid sequence represented by Formula I.

Those applications include, for example, a method for inhibitingthrombus formation in a mammal comprising administering apharmaceutically effective amount of the pharmaceutical composition tothe mammal. The pharmaceutically effective amount may be between about0.001 nM and 1.0 mM, is preferably between about 0.1 nM and 100 μM, andis most preferably between about 1.0 nM and 50 μM. Additionally, thepharmaceutical composition may be administered prior to, following, orsimultaneously with administration of a fibrinolytic or thrombolyticagent such as tissue plasminogen activator, streptokinase, urokinase,prourokinase, and modifications thereof. Alternatively thepharmaceutical composition may be administered in combination with ananticoagulant.

Additionally, other applications include, for example, a method oftreating a mammal for which inhibiting Factor VIIa, Factor XIa, plasmakallikrein, or plasmin is indicated comprising administering apharmaceutically effective amount of the pharmaceutical composition tothe mammal. Such indications include; inflammation, septic shock,hypotension, ARDS, DIC, cardiopulmonary bypass surgery, and bleedingfrom postoperative surgery.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Schematic outline of selected enzymes and mediators thatmodulate the coagulation, contact, fibrinolytic, inflammatory, andcomplement pathways. Activation of these pathways can lead to theclinical states indicated.

FIG. 2. Sequence alignment of Kunitz domains from mammalian sources.Aligned are TF7I-C (residues 1-58) (SEQ ID NO: 33), which is describedherein; APPI (residues 1-58) (SEQ ID NO: 34) from human Alzheimer'sdisease amyloid β-protein precursor, residues 287-344 (Castro, M. et al.FEBS Lett. 267: 207-212 1990!); TFPI-KD1 (residues 22-79) (SEQ ID NO:35), TFPI-KD2 (residues 93-150) (SEQ ID NO: 36), and TFPI-KD3 (residues185-242) (SEQ ID NO: 37) of human TFPI, respectively (Broze Jr., G. J.et al., Biochemistry 29: 7539-7546 1990!); ITI-KD1 and ITI-KD2,(residues 22-79 and 78-135) (SEQ ID NO: 38 and 39) of humaninter-α-trypsin inhibitor, respectively (Vetr, H. et al., FEBS Lett.245: 137-140 1989!); Collagen α 3 (VI) (residues 2899-2956) (SEQ ID NO:40) Collagen alpha 3 (VI) chain precursor (Chu, M. L. et al. EMBO J. 9:385-393 1990!); HKIB9 (7-60) (SEQ ID NO: 41) Human Kunitz-type proteaseinhibitor, HKIB9 (Norris, K., in Genbank Database (Dec. 31, 1993,Release 39.0), submitted Jan. 19, 1994); BPTI (1-58) (SEQ ID NO: 42),Aprotinin, bovine basic pancreatic trypsin inhibitor (Creighton T. E.and Charles, I. G., Cold Spring Harbor Symp. Quant. Biol. 52: 511-5191987!). A motif alignment of invariant residues is listed.

FIG. 3. Model of APPI and other Kunitz domains. The numbers refer to theresidues found in APPI and other Kunitz domains; residue 15 correspondsto the P₁ residue. The shaded area refers to the primary (residues11-19) and secondary (residues 34-39) binding loops of APPI and otherKunitz domains.

FIG. 4. Sequence and Apparent Equilibrium Dissociation Constants forKunitz Domain Variants. Amino acids positions corresponding to those inAPPI are indicated.

FIG. 5. Determination of the Apparent Equilibrium Dissociation Constantsof TF7I-C and APPI with TF-FVIIa. The inhibitory activity is expressedas the fractional activity (inhibited rate/uninhibited rate) at varyinginhibitor concentrations. For this determination, the FVIIa and TF₁₋₂₄₃concentrations were 10 nM and 50 nM, respectively. The apparentequilibrium dissociation constants were determined by nonlinearregression analysis of the data using equation 1 and yielded K_(i) ^(*)values of 2.1 nM for TF7I-C () and 300 nM for APPI (∘). The linesrepresent best fits of the data to equation 1 for the calculated K_(i)^(*). The data is representative of nine independent determinations forTF7I-C and seven for APPI. The fractional activity for 1:1stoichiometric inhibition is represented by a dashed line.

FIG. 6. Prolongation of clotting time in the PT assay in normal humanplasma. The concentration of TF7I-C () and APPI (∘) are plotted vs. thefold prolongation of clotting time upon initiation by TF membranes. Theuninhibited clotting time was 30 sec.

FIG. 7. Prolongation of clotting time in the APTT assay in normal humanplasma. The concentration of TF7I-C () and APPI (∘) are plotted vs. thefold prolongation of clotting time upon initiation by ellagic acid. Theuninhibited clotting time was 31 sec.

DETAILED DESCRIPTION OF THE INVENTION

A. Definitions

Terms used in the claims and specification are defined as set forthbelow unless otherwise specified.

The term amino acid or amino acid residue, as used herein, refers tonaturally-occurring L α-amino acids or residues, unless otherwisespecifically indicated. The commonly used one- and three-letterabbreviations for amino acids are use herein (Lehninger, A. L.,Biochemistry, 2d ed., pp. 71-92, Worth Publishers, N. Y. 1975!, ).

When referring to mutants or variants, the wild type amino acid residueis followed by the residue number and the new or substituted amino acidresidue. For example, substitution of Pro for wild type Thr in residueposition 11 is denominated Thr11Pro.

The P₁ residue refers to the position proceeding the scissile peptidebond of the substrate or inhibitor as defined by Schechter and Berger(Schechter, I. and Berger, A., Biochem. Biophys. Res. Commun. 27:157-162 1967!).

"Expression vector" refers to a DNA construct containing a DNA sequencewhich is operably linked to a suitable control sequence capable ofeffecting the expression of the protein encoded by the DNA in a suitablehost. Such control sequences generally include a promoter to effecttranscription, an optional operator sequence to control transcription, asequence encoding suitable mRNA ribosome binding sites, and sequenceswhich control termination of transcription and translation. The vectormay be a plasmid, a phage particle or "phagemid", or simply a potentialgenomic insert.

Once transformed into a suitable host, the vector may replicate andfunction independently of the host genome, or may, in some instances,integrate into the genome itself. In the present specification,"plasmid", "vector" and "phagemid" are sometimes used interchangeably asthe plasmid is the most commonly used form of vector at present.However, the invention is intended to include such other forms ofexpression vectors which serve equivalent functions and which are, orwhich become, known in the art. "Operably linked," when describing therelationship between two DNA or polypeptide sequences, simply means thatthey are functionally related to each other. For example, a presequenceis operably linked to a peptide if it functions as a signal sequence,participating in the secretion of the mature form of the protein mostprobably involving cleavage of the signal sequence. A promoter isoperably linked to a coding sequence if it controls the transcription ofthe sequence; a ribosome binding site is operably linked to a codingsequence if it is positioned so as to permit translation.

The abbreviations used herein are: TF, tissue factor; FVIIa, FactorVIIa; TFPI, tissue factor pathway inhibitor; ATIII, Antithrombin III;FXa, Factor Xa; FXIa, Factor XIa; FXIIa, Factor XIIa; APPI, Alzheimer'samyloid β-protein precursor inhibitor; TF₁₋₂₄₃, E. coli derivedrecombinant human tissue factor encompassing residues 1-243; BPTI, basicpancreatic trypsin inhibitor; K_(i) ^(*), apparent equilibriumdissociation constant; CHAPS, 3-(3-cholamidopropyl)dimethylammonio!-1-propanesulfonate;)dimethylammoniol!-1-propanesulfonate; PBS, phosphate buffered saline;BSA, bovine serum albumin; HPLC, high performance liquid chromatography;PT, prothrombin time; APTT, activated partial thromboplastin time.

B. Discovery and Preferred Embodiments

The present inventors have discovered that substitutions at certain keyamino acid positions within and around the primary and secondary bindingloops of Kunitz domain serine protease inhibitors can dramaticallyimprove the potency of the inhibitors toward the tissue factor-FVIIacomplex. The present invention therefore provides for polypeptides whichcomprise one or more of these mutant Kunitz domains serine proteaseinhibitors. According to the present invention specific amino acidresidues of APPI and other Kunitz domain proteins are altered to providenovel serine protease inhibitors. The Kunitz domain mutants can comprisea portion of any of a number of proteins to provide a protein that caninhibit tissue factor-FVIIa as well as other serine proteases of thecoagulation cascade. For instance, the Kunitz domain mutants of theinstant invention can replace the Kunitz domain of other proteins knownto have Kunitz domains to provide for novel polypeptides which caninhibit tissue factor-FVIIa as well as other serine proteases of thecoagulation cascade.

According to the present invention, residues 11-19, 34, and 38-39 werealtered by Kunkel mutagenesis as described in Example 1. Variants werethen assayed for their ability to inhibit TF-FVIIa and several otherserine proteases. The best TF-FVIIa Kunitz domain inhibitors showed astrong preference for Arg and Lys in position 15; however proteins withMet at position 15 were also inhibitors. As expected, Gly at position 12and Ala at position 16 produced the best inhibitors (see FIG. 4). Gly isalmost always found at residue 12 in Kunitz domains (Creighton, T. E.and I. G. Charles, Cold Spring Harbor Symp. Quant. Biol. 52: 511-5191987!). Position 16 is usually either Gly or Ala in Kunitz domains(Creighton, T. E. and I. G. Charles, Cold Spring Harbor Symp Quant Biol52: 511-519 1987!). In addition, maintaining Cys at positions 14 and 38generally led to better inhibitors but its requirement was not absolute.

The most preferred residues at other positions in the binding loop werePro at position 11, Leu and Met at position 17, Met and Ile at position18, and Leu or Lys at position 19. However, inhibition of TF-FVIIa wasalso observed when residues Arg, Ala, Glu, Gly, or Thr at position 11,Ile, Arg, Tyr, or Ser at position 17, Leu, Tyr, His, or Phe at position18, and Arg, Ala, or Ile at position 19 were incorporated into theKunitz domain. A number of residues were most preferable at position 34including Val, Ile and Tyr. Other residues here included Leu, Phe, Trp,and Ser. The residues at positions 13 and 39, which are in closeproximity to one another (Hynes, T. R. et al., Biochemistry 29:10018-10022 1990!) (see FIG. 3), also affected TF-FVIIa inhibitoryactivity. Most preferred residues at position 39 included Gly and Tyr;Trp, Phe, and His were also preferred. When Gly was present at position39, the residue at position 13 had a significant effect on TF-FVIIainhibition; large hydrophobic amino acids at position 13 were about4-fold more potent than Pro at this position. When Tyr was present atposition 39, the most potent inhibitors were found (K_(i) ^(*) =2-3 nM)and the residue at position 13 made little difference in bindingaffinity. Therefore, with respect to potent inhibition of TF-FVIIa,hydrophobic residues at either or both positions 13 and 39 arepreferred; elimination of these favorable interactions leads to lesspotent inhibitors.

Several inhibitors in FIG. 4 represent Kunitz domain inhibitors that areable to potently inhibit TF-FVIIa in the absence of FXa. For example,the TF7I-C variant differs by 4 key amino acids (Thr11Pro, Met17Leu,Ser19Leu and Gly39Tyr) compared to wild type APPI and results in anincrease in affinity for the TF-FVIIa complex of greater than 150-fold.BPTI has recently been shown to competitively inhibit TF-FVIIa activity,albeit with relatively weak affinity (K_(i) =30 82 M) (Chabbat, J. etal., Thromb Res 71: 205-215 1993!). In addition, the first Kunitz domainof TFPI itself does not potently inhibit TF-FVIIa, having a K_(i) ^(*)of ca. 600 nM.

The increased affinity of TF7I-C for TF-FVIIa relative to APPI isreflected in its ability to prolong the clotting time in a prothrombintime assay (FIG. 6). At 40 μM, APPI had little effect (1.5-foldprolongation), whereas TF7I-C prolonged the clotting time initiated byTF membranes by 3.5-fold. BPTI has recently been shown to inhibitTF-induced coagulation; however, ca. 75 μM was needed to prolong theclotting time 1.4-fold in a PT assay (van den Besselaar, A. M. H. P. etal., Thromb Haemostas 69: 298-299 1993!).

Interestingly, many of the inhibitors that resulted in increased bindingaffinity for TF-FVIIa, also resulted in more potent inhibitors of FXIaand kallikrein. This implies that the active sites of FVIIa and FXIa aresomewhat alike, which may not be too surprising since Factor IX is asubstrate for both proteases. APPI is a potent inhibitor of FXIa(Wagner, S. L. et al., Biochem. Biophys. Res. Commun. 186: 1138-11451992!). Inhibition of kallikrein was unexpected and suggests that theactive site of kallikrein may also be similar. In contrast, TF7I-C, forexample, did not inhibit thrombin, Factor XIIa or activated protein C(Ki*>10 μM) and only poorly inhibited FXa (Ki*=90 nM) and plasmin(Ki*=40±6 nM). TF7I-C is much more effective than APPI at prolonging theclotting time in the APTT assay, a measure of the intrinsic coagulationpathway (FIG. 7). This is consistent with more potent inhibition of FXIaand kallikrein by TF7I-C compared to APPI. Much higher concentrations ofinhibitor (>100 Ki*) are required to prolong clotting times in theplasma assays compared to the conditions required for potent inhibitionin vitro. Possible explanations include suboptimal binding conditionsprovided in the in vitro clotting assays, solution vs. membrane boundinhibition of proteases, kinetics of inhibition, and cross reactivitywith other plasma serine proteases.

On the other hand, several inhibitors in FIG. 4 resulted in specificinhibition of TF-FVIIa with Ki*>100 nM for FXIa and kallikrein. Theseinhibitors generally had Lys present at position 19 and Met at position18. At position 19, Lys or Arg are preferred for specific inhibition ofTF-FVIIa. Although their ability to inhibit FXIa and kallikrein has beenreduced, they are still potent inhibitors of TF-FVIIa (see FIG. 4).

According to the present invention, residues 20 and 46, as well asresidue 44, in addition to the putative contact residues in the primarybinding loop (residues 11-19) and secondary binding loop (residues 34,38 and 39), (see FIG. 2) of APPI have been identified as key residues.In addition to information derived from the x-ray crystal structures ofserine proteases with Kunitz domain inhibitors, the amino acid sequencesof trypsin and the serine protease domain of FVIIa can be aligned tostudy the potential interactions between FVIIa and APPI. The variableregion 2 (residues 59-62; chymotrypsinogen numbering system, see, forexample, Greer, J., (1990) Proteins 7:317-334) in FVIIa contains 8residues compared to only 3 in trypsin. Without being limited to any onetheory, this may allow this surface loop to make contact with a boundKunitz domain. In FVIIa, residue 59b of this loop is a Lys, which,without limitation to a theory, may sterically overlap with residueArg20 in APPI. This is an unfavorable interaction (both sterically andelectrostatically). In the APPI crystal structure, the surface residue,Arg20 is remarkably well defined probably due to the packing of otherside chains in APPI, suggesting, without limitation to one theory, thatrotation to avoid collision with Lys59b in FVIIa is improbable. Withoutreliance on any one theory, residue 59b in FVIIa, in contrast, canprobably freely rotate which may put it in favorable contact with Asp46of APPI or an appropriate residue at position 18 in APPI. In addition,position 18 of APPI is also near FVIIa (Lys59b) and APPI (Arg20) and mayplay a role in these binding interactions.

Based on these observations, residues 20 and 44, and 46 of APPI Kunitzdomain or a polypeptide as described herein are modified. According tothis aspect of the present invention Arg20 may be modified to Ala, Val,Ser, Thr, Asn, Gin, Asp, Glu, Leu, or Ile. At residue 46 any hydrogenbond acceptor may be substituted for Asp. In one embodiment either Aspor Glu are found at position 46. Residues at position 44 as well asposition 20 and 46, as well as the residues of the primary and secondarybinding loops are all meant to promote favorable interactions betweenKunitz domain proteins and the tissue factor-Factor VIIa complex. Suchfavorable interaction can be assessed according to those assaysdescribed herein for the measurement of apparent K_(i) ^(*) with respectto tissue factor-Factor VIIa.

C. Utility

As previously indicated, many common human disorders arecharacteristically associated with a hypercoagulable state leading tointravascular thrombi and emboli (Thrombosis in CardiovascularDisorders, (Fuster, V. and Verstraete, M., eds.), W. B. Saunders,Philadelphia 1992!). These are a major cause of medical morbidity,leading to phlebitis, infarction, and stroke, and of mortality, fromstroke and pulmonary and cardiac emboli. A large percentage of suchpatients have no antecedent risk factors, and develop venousthrombophlebitis and subsequent pulmonary emboli without a known cause.Other patients who form venous thrombi have underlying diseases known topredispose to these syndromes.

Some of these patients may have genetic or acquired deficiencies offactors that normally prevent hypercoagulability, such as antithrombinIII. Others have mechanical obstructions to venous flow, such as tumormasses, that lead to low flow states and thrombosis. Patients withmalignancy have a high incidence of thrombotic phenomena, for unclearreasons. Antithrombotic therapy in this situation with currentlyavailable agents is dangerous and often ineffective.

Patients with atherosclerosis are predisposed to arterial thromboembolicphenomena for a variety of reasons. Atherosclerotic plaques form nidusesfor platelet plugs and thrombi that lead to vascular narrowing andocclusion, resulting in myocardial and cerebral ischemic disease.Thrombi that break off and are released into the circulation can causeinfarction of different organs, especially the brain, extremities, heartand kidneys. After myocardial infarctions, clots can form in weak,poorly functioning cardiac chambers and be released into the circulationto cause emboli. All such patients with atrial fibrillation are felt tobe at great risk for stroke and require antithrombotic therapy.

In addition, thrombolytic therapy for acute myocardial infarction hasbecome an established procedure for patients (Collen, D. and Stump, D.,Ann Rev Med .39:405-423 1988!). However, currently availablethrombolytic agents are not effective in all patients which is manifestby reocclusion, resistance to reperfusion, prolonged times to achievenormal coronary flow and the like.

Patients whose blood flows over artificial surfaces, such as prostheticsynthetic cardiac valves or hip replacements, or through extracorporealperfusion devices, are also at risk for the development of plateletplugs, thrombi, and emboli. It is standard practice that patients withartificial cardiac valves be chronically anti-coagulated.

Thus, a large category of patients, including those with cancer,atherosclerosis, coronary artery disease (PTCA, CABG, Post MI, etc.),unstable angina, artificial heart valves, and a history of stroke,transient ischemic attacks, atrial fibrillation, deep vein thrombosis,phlebitis, or pulmonary emboli, are candidates for limited or chronicantithrombotic therapy. However, this therapy is often ineffective ormorbid in its own right. This is partially because the number ofavailable therapeutic agents is limited. Available antiplatelet agents,such as aspirin, inhibit the cyclooxygenase-induced activation ofplatelets only and are often inadequate for therapy. Availableanticoagulants include heparin and warfarin which are not alwaysefficacious and can often have side effects including increased bleedingrisk and problems associated with monitoring these therapies.

An agent which effectively inhibits the formation of fibrin fromfibrinogen should accordingly be particularly useful in therapeuticintervention in a large group of disorders characterized by ahypercoagulable state.

As a general matter, however, in the management of thromboembolic andinflammatory disorders, the compounds of the present invention may beutilized in compositions with a pharmaceutically acceptable excipientfor injectable administration, in compounds such as tablets, capsules,or elixirs for oral administration. Animals in need of treatment usingcompounds of the present invention can be administered dosages that willprovide optimal efficacy. The dose and method of administration willvary from animal to animal, and be dependent upon such factors asweight, diet, concurrent medication, and other factors which thoseskilled in the medical arts will recognize.

There are many approaches to the regulation of blood coagulation rangingfrom the currently used nonspecific inhibitors warfarin and heparin, tomore selective agents, such as thrombin or FXa inhibitors. FVIIa, FXIa,and plasma kallikrein represent other targets for regulation of bloodcoagulation since they can either initiate or contribute to coagulationby the extrinsic or intrinsic pathway. The zymogens are present inplasma at lower concentrations than prothrombin and their effects uponhemostasis are considerably amplified. Thus inhibitors of these enzymesshould be very potent, since any uninhibited enzyme can contribute tofurther activation of the cascade. The current anticoagulant therapies(heparin, warfarin) are nonspecific inhibitors. Specific inhibitors maybe advantageous with respect to side effects such as bleeding. On theother hand coordinate inhibition of several proteases may be desirablefor certain indications. The regulation of coagulation and itsrelationship to disease is a very complex process.

TF-FVIIa is an appropriate target for the intervention in coagulationprocesses because it is thought to initiate the cascade (see FIG. 1)(Broze Jr., G. J. et al., Biochemistry 29: 7539-7546 1990; Broze Jr., G.J., Semin. Hematol.29: 159-169 1992!). Thus, the inhibition of FVIIa,FXIa, and/or plasma kallikrein by agents described herein represents anapproach for clinical intervention in various thrombotic disorders. Thusthe agents described herein are useful in the treatment of thrombosis.More specifically, the instant inhibitors are especially useful asadjunct therapy for thrombolysis, unstable angina, deep vein thrombosis,hip replacement, coronary artery bypass graft, percutaneous transluminalcoronary angioplasty, pulmonary embolism, septic shock, and DIC.

The agents described herein are also useful in the treatment of diseaseswhere intervention in the activation of the contact pathway orneutrophil activation is indicated (e.g. inflammation, coagulation,fibrinolysis, and complement activation). More specifically, the instantinhibitors are especially useful in the treatment of diseases whereinhibition of FXIa, kallikrein, FXIIa, FXa, and HLE, complement isindicated (see FIG. 1) as for example in the treatment of sepsis orseptic shock, inflammation, ARDS, DIC, hypotension, cardiopulmonarybypass surgery, and for bleeding from postoperative surgery.

The agents described herein may be useful in clinical situations thatrequire acute or chronic therapy. It is anticipated that indications forwhich acute therapy is indicated are more preferred than those forchronic therapy. The pharmaceutical use of foreign or mutant humanproteins may be immunogenic; however foreign proteins are used to treatacute indications. An example of such a protein is streptokinase, aprotein derived from streptococci that acts as a fibrinolytic and iscommonly used to treat acute myocardial infarction. The agents describedherein may elicit an immune response; however related foreign proteinssuch as BPTI have been used in humans clinically and are not anticipatedto elicit a serious immune response. The covalent attachment ofpolyethylene glycol (PEG) to the agents described herein may reduce theimmunogenicity and toxicity, and prolong the half-life as has beenobserved with other proteins (Katre N. V., J. Immunol. 144:209-2131990!; Poznansky, M. J. et al., FEB 239:18-22 1988!; Abuchowski, A. etal., J. Biol. Chem. 252:3582-3586 1977!)

D. Methods of Making

Chemical Synthesis

One method of producing the Kunitz domain polypeptides of Formula Iinvolves chemical synthesis of the protein, followed by treatment underoxidizing conditions appropriate to obtain the native conformation, thatis, the correct disulfide bond linkages. This can be accomplished usingmethodologies well known to those skilled in the art (see Kelley, R. F.and Winkler, M. E. in Genetic Engineering Principles and Methods,(Setlow, J. K., ed.)., Plenum Press, N.Y., vol. 12, pp. 1-19 1990!;Stewart, J. M. and Young, J. D. Solid Phase Peptide Synthesis PierceChemical Co. Rockford, Ill. 1984!).

Polypeptides of the invention, especially those containing 58 amino acidresidues or fewer, may be prepared using solid phase peptide synthesis(Merrifield, J. Am. Chem. Soc., 85:2149 1964!; Houghten, Proc. Nati.Acad. Sci. USA 82:5132 1985!). Solid phase synthesis begins at thecarboxy-terminus of the putative peptide by coupling a protected aminoacid to a suitable resin, as shown in FIGS. 1-1 and 1-2, on pages 2 and4 of Stewart and Young supra.

In synthesizing polypeptides of this invention, the carboxyl terminalamino acid, with its α-amino group suitably protected, is coupled to achloromethylated polystyrene resin (see FIG. 1-4, page 10 of Stewart andYoung supra.). After removal of the α-amino protecting group with, forexample, trifluoroacetic acid (TFA) in methylene chloride andneutralizing in, for example TEA, the next cycle in the synthesis isready to proceed.

The remaining α-amino- and, if necessary, side-chain-protected aminoacids are then coupled sequentially in the desired order by condensationto obtain an intermediate compound connected to the resin.Alternatively, some amino acids may be coupled to one another forming apeptide prior to addition of the peptide to the growing solid phasepolypeptide chain.

The condensation between two amino acids, or an amino acid and apeptide, or a peptide and a peptide can be carried out according to theusual condensation methods such as azide method, mixed acid anhydridemethod, DCC (dicyclohexylcarbodiimide) method, active ester method(p-nitrophenyl ester method, BOP benzotriazole-1-yl-oxy-tris(dimethylamino) phosphonium hexafluorophosphate! method,N-hydroxysuccinic acid imido ester method, etc.), and Woodward reagent Kmethod. In the case of elongating the peptide chain in the solid phasemethod, the peptide is attached to an insoluble carrier at theC-terminal amino acid. For insoluble carriers, those which react withthe carboxy group of the C-terminal amino acid to form a bond which isreadily cleaved later, for example, halomethyl resin such aschloromethyl resin and bromomethyl resin, hydroxymethyl resin,aminomethyl resin, benzhydrylamine resin, andt-alkyloxycarbonyl-hydrazide resin can be used.

Common to chemical syntheses of peptides is the protection of thereactive side-chain groups of the various amino acid moieties withsuitable protecting groups at that site until the group is ultimatelyremoved after the chain has been completely assembled. Also common isthe protection of the α-amino group on an amino acid or a fragment whilethat entity reacts at the carboxyl group followed by the selectiveremoval of the a-amino-protecting group to allow subsequent reaction totake place at that location. Accordingly, it is common that, as a stepin the synthesis, an intermediate compound is produced which includeseach of the amino acid residues located in the desired sequence in thepeptide chain with various of these residues having side-chainprotecting groups. These protecting groups are then commonly removedsubstantially at the same time so as to produce the desired resultantproduct following purification.

The applicable protective groups for protecting the α-and ε-amino sidechain groups are exemplified by benzyloxycarbonyl (abbreviated Z),isonicotinyloxycarbonyl (iNOC), O-chlorobenzyloxycarbonyl Z (NO₂ !,p-methoxybenzyloxycarbonyl Z(OMe)!, t-butoxycarbonyl, (Boc),t-amyioxycarbonyl (Aoc), isobornyloxycarbonyl, adamatyloxycarbonyl,2-(4-biphenyl)-2propyloxycarbonyl (Bpoc), 9-fluorenylmethoxycarbonyl(Fmoc), methylsulfonyiethoxycarbonyl (Msc), trifluoroacetyl, phthalyl,formyl, 2-nitrophenylsulphenyl (NPS), diphenylphosphinothioyl (Ppt),dimethylophosphinothioyl (Mpt) and the like.

As protective groups for carboxy group there can be exemplified, forexample, benzyl ester (OBzl), cyclohexyl ester (Chx), 4-nitrobenzylester (ONb), t-butyl ester (Obut), 4-pyridylmethyl ester (OPic), and thelike. It is desirable that specific amino acids such as arginine,cysteine, and serine possessing a functional group other than amino andcarboxyl groups are protected by a suitable protective group as occasiondemands. For example, the guanidino group in arginine may be protectedwith nitro, p-toluenesulfonyl, benzyloxycarbonyl, adamantyloxycarbonyl,p-methoxybenzenesulfonyl, 4-methoxy-2, 6-dimethylbenzenesulfonyl (Mds),1,3,5-trimethylphenysulfonyl (Mts), and the like. The thiol group incysteine may be protected with p-methoxybenzyl, triphenylmethyl,acetylaminomethyl ethylcarbamoyl, 4-methylbenzyl, 2, 4,6-trimethy-benzyl (Tmb) etc, and the hydroxyl group in the serine can beprotected with benzyl, t-butyl, acetyl, tetrahydropyranyl etc.

Stewart and Young supra provides detailed information regardingprocedures for preparing peptides. Protection of α-amino groups isdescribed on pages 14-18, and side-chain blockage is described on pages18-28. A table of protecting groups for amine, hydroxyl and sulfhydrylfunctions is provided on pages 149-151.

After the desired amino acid sequence has been completed, theintermediate peptide is removed from the resin support by treatment witha reagent, such as liquid HF and one or more thio-containing scavengers,which not only cleaves the peptide from the resin, but also cleaves allthe remaining side-chain protecting groups. Following HF cleavage, theprotein sequence is washed with ether, transferred to a large volume ofdilute acetic acid, and stirred at pH adjusted to about 8.0 withammonium hydroxide.

Preferably in order to avoid alkylation of residues in the polypeptide,(for example, alkylation of methionine, cysteine, and tyrosine residues)a thiocresol and cresol scavenger mixture is used. The resin is washedwith ether, and immediately transferred to a large volume of diluteacetic acid to solubilize and minimize intermolecular cross-linking. A250 μM polypeptide concentration is diluted in about 2 liters of 0.1 Macetic acid solution. The solution is then stirred and its pH adjustedto about 8.0 using ammonium hydroxide. Upon pH adjustment, thepolypeptide takes its desired conformational arrangement.

Kunitz domains can be made either by chemical synthesis, describedabove, or by semisynthesis. The chemical synthesis or semisynthesismethods of making allow the possibility of non-natural amino acidresidues to be incorporated. This has been carried out for Kunitzdomains and related proteins as previously described (Beckmann, J. etal., Eur. J. Biochem. 176: 675-682 1988!; Bigler, T. L. et al., Prot.Sci. 2: 786-799 1993!).

Gene Synthesis, Cloning, and Expression

General Procedures From the amino acid sequence, as provided in FormulaI, the purified protein may be produced using standard recombinant DNAtechniques. These techniques contemplate, in simplified form, taking agene encoding the Kunitz domain polypeptides of Formula I; inserting itinto an appropriate vector; inserting the vector into an appropriatehost cell; culturing the host cell to cause expression of the Kunitzdomain polypeptides of Formula I; and purifying the protein producedthereby.

Somewhat more particularly, the DNA sequence encoding the Kunitz domainpolypeptides of Formula I is cloned and manipulated so that it may beexpressed in a convenient host. DNA encoding Formula I polypeptides canbe obtained by synthetically constructing the DNA sequence (Sambrook, J.et al,, Molecular Cloning (2nd ed.), Cold Spring Harbor Laboratory, N.Y.1989!).

The DNA encoding Formula I peptides is then inserted into an appropriateplasmid or vector which is used to transform a host cell. In general,plasmid vectors containing replication and control sequences which arederived from species compatible with the host cell are used inconnection with those hosts. The vector ordinarily carries a replicationsite, as well as sequences which encode proteins that are capable ofproviding phenotypic selection in transformed cells.

For example, E. coli may be transformed using pBR322, a plasmid derivedfrom an E. coli species (Mandel, M. et al., J. Mol. Biol. 53:154 1970!).Plasmid pBR322 contains genes for ampicillin and tetracyclineresistance, and thus provides easy means for selection. Other vectorsinclude different features such as different promoters, which are oftenimportant in expression. For example, plasmids pKK223-3, pDR720, andpPL-lambda represent expression vectors with the tac, trp, or P_(L)promoters that are currently available (Pharmacia Biotechnology).

Direct expression of the Kunitz domain polypeptides of Formula I

A preferred vector is pSAlz1. This vector was created as described inExample 1 and contains origins of replication for E. coli, the alkalinephosphatase promoter, the stII signal sequence and APPI variant gene,and the ampicillin resistance gene. Other preferred vectors are pBO475,pR1T5 and pR1T2T (Pharmacia Biotechnology). These vectors containappropriate promoters followed by the Z domain of protein A, allowinggenes inserted into the vectors to be expressed as fusion proteins.Further discussion of these vectors may be found below.

Other preferred vectors can be constructed using standard techniques bycombining the relevant traits of the vectors described herein. Relevanttraits of the vector include the promoter, the ribosome binding site,the APPI variant gene or gene fusion (the Z domain of protein A and APPIvariant and its linker), the signal sequence, the antibiotic resistancemarkers, the copy number, and the appropriate origins of replication.

In E. coli, Kunitz domains have been expressed as intact secretedproteins (Castro, M. et al., FEBS Lett. 267: 207-212 1990!),intracellularly expressed proteins (Altman, J. D. et al., Protein Eng.4: 593-600 1991!), or as fusion proteins (Sinha, S. et al., J. Biol.Chem. 266: 21011-21013 1991!; Lauritzen, C. et al., Prot. Express.Purif. 2: 372-378 1991!; Auerswald, E. A. et al ., Biol. Chem.Hoppe-Seyler 369: 27-35 1988!).

The host cell may be prokaryotic or eukaryotic. Prokaryotes arepreferred for cloning and expressing DNA sequences to produce parentpolypeptides, segment substituted polypeptides, residue-substitutedpolypeptides and polypeptide variants. For example, E. coli K12 strain294 (ATCC No. 31446) may be used as E. coli B, E. coli X1776 (ATCC No.31537), and E. coli c600 and c600hfl, E. coli W3110 (F-, gamma-,prototrophic /ATCC No. 27325), bacilli such as Bacillus subtilis, andother enterobacteriaceae such as Salmonella -typhimurium or Serratiamarcesans, and various pseudomonas species. The preferred prokaryote isE. coli W3110 (ATCC 27325). When expressed by prokaryotes thepolypeptides typically contain an N-terminal methionine or a formylmethionine and are not glycosylated. In the case of fusion proteins, theN-terminal methionine or formyl methionine resides on the amino terminusof the fusion protein or the signal sequence of the fusion protein.These examples are, of course, intended to be illustrative rather thanlimiting.

In addition to prokaryotes, eukaryotic organisms, such as yeastcultures, or cells derived from multicellular organisms may be used. Inprinciple, any such cell culture is workable. However, interest has beengreatest in vertebrate cells, and propagation of vertebrate cells inculture (tissue culture) has become a reproducible procedure (TissueCulture, Academic Press, Kruse and Patterson, eds. 1973!). Examples ofsuch useful host cell lines are VERO and HeLa cells, Chinese HamsterOvary (CHO) cell lines, W138, 293, BHK, COS-7 and MDCK cell lines. Yeastexpression systems have been used to make Kunitz domains (Wagner, S. L.et al., Biochem. Biophys. Res. Commun. 186: 1138-1145 1992!; Vedvick, T.et al., J. Indust. Microbiol. 7: 197-202 1991!). In particular the yeastPichia pastoris has been used successfully using the Saccharomycescerevisiae α mating factor prepro signal sequence and the P. pastorisalcohol oxidase AOX1 promoter and terminator sequences. Other yeastexpression vectors and hosts commonly used to express heterologousproteins are also contemplated.

Gene Fusions

A variation on the above procedures contemplates the use of genefusions, wherein the gene encoding the APPI variant is associated, inthe vector, with a gene encoding another protein or a fragment ofanother protein. This results in the APPI variant being produced by thehost cell as a fusion with another protein. The "other" protein is oftena protein or peptide which can be secreted by the cell, making itpossible to isolate and purify the desired protein from the culturemedium and eliminating the necessity of destroying the host cells whicharises when the desired protein remains inside the cell. Alternatively,the fusion protein can be expressed intracellularly. It is useful to usefusion proteins that are highly expressed.

The use of gene fusions, though not essential, can facilitate theexpression of heterologous proteins in E. coli as well as the subsequentpurification of those gene products (Harris, T. J. R. in GeneticEngineering, (Williamson, R., ed.), Academic, London, Vol. 4, p. 1271983!; Uhlen, M. and Moks, T., Methods Enzymol. 185:129-143 1990!).Protein A fusions are often used because the binding of protein A, ormore specifically the Z domain of protein A, to IgG provides an"affinity handle" for the purification of the fused protein (Nilsson, B.and Abrahmsen, L. Methods Enzymol. 185:144-161 1990!). It has also beenshown that many heterologous proteins are degraded when expresseddirectly in E. coli, but are stable when expressed as fusion proteins(Marston, F. A. O., Biochem J. 240: 1 1986!).

APPI variants expressed as fusion proteins may be properly folded or mayrequire folding to obtain the native structure. The properly foldedfusion protein may be active and useful as a serine protease inhibitor.More preferred would be the correctly folded native protein that isobtained from the fusion protein by methods known in the art. Fusionproteins can be cleaved using chemicals, such as cyanogen bromide, whichcleaves at a methionine, or hydroxylamine, which cleaves between an Asnand Gly. Using standard recombinant DNA methodology, the nucleotide basepairs encoding these amino acids may be inserted just prior to the 5'end of the APPI variant gene.

Alternatively, one can employ proteolytic cleavage of fusion proteins,which has been recently reviewed (Carter, P. in Protein Purification:From Molecular Mechanisms to Large-Scale Processes, (Ladisch, M. R.,Willson, R. C., Painton, C. C., and Builder, S. E., eds.), AmericanChemical Society Symposium Series No. 427, Ch 13, pp. 181-193 1990!).

Proteases such Factor Xa, thrombin, subtilisin and mutants thereof, havebeen successfully used to cleave fusion proteins. Typically, a peptidelinker that is amenable to cleavage by the protease used is insertedbetween the "other" protein (e.g., the Z domain of protein A) and theprotein of interest, such as an APPI variant. Using recombinant DNAmethodology, the nucleotide base pairs encoding the linker are insertedbetween the genes or gene fragments coding for the other proteins.Proteolytic cleavage of the partially purified fusion protein containingthe correct linker can then be carried out on either the native fusionprotein, or the reduced or denatured fusion protein.

The protein may or may not be properly folded when expressed as a fusionprotein. Also, the specific peptide linker containing the cleavage sitemay or may not be accessible to the protease. These factors determinewhether the fusion protein must be denatured and refolded, and if so,whether these procedures are employed before or after cleavage.

When denaturing and refolding are needed, typically the protein istreated with a chaotrope, such a guanidine HCl, and is then treated witha redox buffer, containing, for example, reduced and oxidizeddithiothreitol or glutathione at the appropriate ratios, pH, andtemperature, such that the protein of interest is refolded to its nativestructure.

Mutant DNA Production

As previously discussed, various techniques are also available which maynow be employed to produce mutant APPI DNA, which encodes for additions,deletions, or changes in amino acid sequence of the resultant proteinrelative to the parent APPI molecule.

By way of illustration, with expression vectors encoding APPI in hand,site specific mutagenesis (Kunkel et al., Methods Enzymol. 204:125-1391991!; Carter, P., et al., Nucl. Acids. Res. 13:4331 1986!; Zoller, M.J. et al., Nucl. Acids Res. 10:6487 1982!), cassette mutagenesis (Wells,J. A., et al., Gene 34:315 1985!), restriction selection mutagenesis(Wells, J. A., et al., Philos. Trans, R. Soc. London SerA 317, 4151986!) or other known techniques may be performed on the APPI DNA. Themutant DNA can then be used in place of the parent DNA by insertion intothe aforementioned expression vectors. Growth of host bacteriacontaining the expression vectors with the mutant DNA allows theproduction of mutant APPI (i.e., analogs or homologs of APPI), which canbe isolated as described herein.

Purification and characterization

Purification and characterization of APPI variants may be carried out byany art standard technique including gel filtration, ion exchange,hydrophobic interaction, and affinity chromatography. In the instantcase, recombinant APPI variants were purified from the media of E. coligrown in 10 l fermentors or shake flasks by chromatography on a trypsinaffinity column followed by reverse phase C18 HPLC.

Following site-directed mutagenesis of the APPI gene and confirmation ofclones by DNA sequence analysis, variant proteins were expressed in andpurified from E. coli. The Kunitz domains were concentrated andpartially purified from the media using a trypsin affinity column. Thefinal purification was carried out using reverse phase C18 HPLC asdescribed in Example 2. The expression level of most of the variants wasca. 1 mg/L in shake flasks and 80-100 mg/L in 10 L fermentations.Following purification, protein sequences were verified by massspectrometry for the correct mass predicted from the sequence, assumingthat all three disulfides were formed; all were within the error of thismeasurement (±2 amu). HPLC chromatographs of inhibitors containing a Metcommonly displayed a small peak eluting just before or just after themajor inhibitor peak. This was a result of methionine oxidation to thesulfoxide.

E. Methods of Analysis

Apparent equilibrium dissociation constants (K_(i) ^(*)) were determinedusing methods derived for tight-binding inhibitors (Bieth, J.,Proteinase Inhibitors 463-469 1974!; Williams, J. W. and Morrison, J.F., Methods Enzymol 63: 437-467 1979!), assuming enzyme and inhibitorform a reversible complex with a 1:1 stoichiometry as has been observedfor the interaction of Kunitz domains with serine proteases (Bode, W.and R. Huber, Eur. J. Biochem. 204: 433-451 1992; Laskowski, M., Jr. andI. Kato, Annu. Rev. Biochem. 49: 593-626 1980!). The data were fit bynonlinear regression analysis to Equation 1: ##EQU1## where V_(i) /V_(o)is the fractional activity (steady-state inhibited rate divided by theuninhibited rate), E_(o) ! is the total FVIIa active site concentration,and I_(o) ! is the total inhibitor concentration. Variants were assayedfor their binding affinity to TF-FVIIa and those with K_(i) ^(*) valuesranging from ca. 1-500 nM are shown in FIG. 4. The inhibition ofTF-FVIIa by TF7I-C and APPI under equilibrium conditions is shown inFIG. 5; apparent K_(i) ^(*) values of 1.9±0.4 nM and 301±44 nM werecalculated for TF7I-C and APPI with TF-FVIIa, respectively (FIG. 4).

By measuring apparent K_(i) ^(*) values with other relevant serineproteases found in human plasma, the relative specificities of wild typeAPPI, TF7I-C, and other mutant inhibitors were determined. To aliquotsof serial diluted inhibitor, either activated protein C, thrombin, FXa,FXIa, FXIIa, plasma kallikrein or plasmin were added. After incubationand addition of the appropriate substrate, plots of fractional activityversus inhibitor concentration were generated as described in Example 3.Apparent equilibrium dissociation constants (K_(i) ^(*)) were calculatedfrom equation (1) and are reported in FIG. 4. APPI was a potentinhibitor of FXIa, in good agreement with previously reported results(Wagner, S. L. et al., Biochem. Biophys. Res. Commun. 186: 1138-11451992!) and a moderate inhibitor of TF-FVIIa, plasmin, and plasmakallikrein; the K_(i) ^(*) for activated protein C, thrombin, FXa, orFXIIa was >10 μM. In addition to potently inhibiting TF-FVIIa, TF7I-C isalso a potent inhibitor of FXIa and plasma kallikrein and a moderateinhibitor of plasmin (FIG. 4). The K_(i) ^(*) for FXa=90 nM and was >10μM for activated protein C, thrombin, or FXIIa. Other inhibitors weremore specific inhibitors of TF-FVIIa with respect to FXIa, plasmakallikrein, or plasmin (FIG. 4).

Based on a tissue factor initiated prothrombin time (PT) assay describedin Example 4, both TF7I-C and APPI prolonged the clotting time in aconcentration dependent manner (FIG. 6). This is consistent with theability of these inhibitors to prevent FX activation through inhibitionof the TF-FVIIa complex. In this assay TF7I-C prolonged the clottingtime 3.5-fold at ca. 40 μM, whereas the same concentration of APPIresulted in only a 1.5-fold increase in the clotting time. TF7I-C alsoshowed concentration dependent inhibition of the surface mediatedcontact activation pathway, as measured by the activated partialthromboplastin time assay (APTT) described in Example 4, a greater than10-fold prolongation of the clotting time at ca. 7 μM was observed (FIG.7). APPI was somewhat less potent in the APTT relative to TF7I-C, havinga clotting time of ca. 3-fold at the same concentration.

F. Pharmaceutical Compositions

Dosage formulations of the compounds of the present invention to be usedfor therapeutic applications must be sterile. Sterility is readilyaccomplished by filtration through sterile filtration membranes such as0.2 μ membranes. Protein formulations ordinarily will be stored inlyophilized form or as an aqueous solution. The pH of the proteinpreparations typically will be between about 3 and 11, more preferablyfrom about 5 to 9, and most preferably from about 7 to 8. The preferredroute of administration is by hypodermic needle.

Therapeutic protein formulations are generally placed into a containerhaving a sterile access port, for example, an intravenous solution bagor vial having a stopper pierceable by a hypodermic injection needle.

Therapeutically effective dosages may be determined by either in vitro(see assays above) or in vivo methods. Based upon such assay techniques,a therapeutically effective dosage range may be determined. The range oftherapeutically effective dosages will naturally be affected by theroute of administration. For injection by hypodermic needle, it may beassumed that the dosage is delivered into the body's fluids. For otherroutes of administration, the adsorption efficiency must be individuallydetermined for APPI variants by methods well-known in pharmacology.

The range of therapeutic dosages may range from about 0.001 nM to about1.0 mM, more preferably from about 0.1 nM to about 100 μM, and mostpreferably from about 1.0 nM to about 50 μM.

A typical formulation of APPI variants as a pharmaceutical compositioncontains from about 0.5 to 500 mg of a compound or mixture of compoundsas either the free acid or base form or as a pharmaceutically acceptablesalt. These compounds or mixtures are then compounded with aphysiologically acceptable vehicle, carrier, excipient, binder,preservative, or stabilizer, etc., as called for by acceptedpharmaceutical practice. The amount of active ingredient in thesecompositions is such that a suitable dosage in the range indicated isobtained.

Sterile compositions for injection can be formulated according toconventional pharmaceutical practice. For example, dissolution orsuspension of the active compound in a vehicle such as water ornaturally occurring vegetable oil like sesame, peanut, or cottonseed oilor a synthetic fatty vehicle like ethyl oleate or the like may bedesired. Buffers, preservatives, antioxidants and the like can beincorporated according to accepted pharmaceutical practice.

The present invention has of necessity been discussed herein byreference to certain specific methods and materials. It is to beunderstood that the discussion of these specific methods and materialsin no way constitutes any limitation on the scope of the presentinvention, which extends to any and all alternative materials andmethods suitable for accomplishing the ends of the present invention.

EXAMPLES Materials

Human Factor VIIa, Factor Xa, Factor XIa, activated protein C andthrombin were purchased from Haematologic Technologies Inc. (Essex Jct.,Vt.). Human plasma kallikrein and Factor XIIa were purchased from EnzymeResearch Laboratories, Inc. (South Bend, Ind.). Recombinant human tissuefactor₁₋₂₄₃ (TF₁₋₂₄₃) was produced in E. coli and purified as previouslydescribed (Paborsky, L. R. et al., Biochemistry 28: 8072-8077 1989!;Paborsky, L. R. et al., J. Biol. Chem. 266: 21911-21916 1991!). Bovinetrypsin, 4-methylumbelliferyl p-guanidinobenzoate and CHAPS werepurchased from Sigma Chemicals, Inc. Bovine serum albumin (BSA),Fraction V was obtained from Calbiochem (La Jolla, Calif.) . N.sup.α-Benzoyl-L-arginine-p-nitroanilide was purchased from Bachem California(Torrance, Calif.). Human plasmin, S-2302, S-2251 and S-2366 werepurchased from Kabi Vitrum (Sweden) and Spectrozyme fXa was purchasedfrom American Diagnostica (Greenwich, Conn.). Affigel-10 was obtainedfrom Bio-Rad Laboratories (Richmond, Calif.). All other reagents wereobtained were of the highest grade commercially available.

Example 1 Plasmid Construction and Mutagenesis

The plasmid pSAlz1 was constructed by inserting a synthetic geneencoding the APPI sequence into an appropriate expression vector forsecretion of APPI into the periplasm and media. The pSAlzl vectorcontained the alkaline phosphatase promoter, stII secretion signal, theAPPI gene, the f1 and colE1 origins of replication, and the ampicillinresistance gene as described by Castro et al. (Castro, M. et al., FEBSLett. 267: 207-212 1990!). The construction of APPI mutants using thepSAlzl vector was accomplished using site-directed oligonucleotidemutagenesis in as previously described (Kunkel, T. A. et al., MethodsEnzymol. 204: 125-139 1991!); selected clones were analyzed by dideoxysequence analysis (Sanger, F. et al., Proc Natl Acad Sci USA 74:5463-5467 1977!).

Example 2 Inhibitor Expression, Purification, and Characterization

Phagemids encoding either APPI or the selected mutants were transformedinto E. coli strain 27C7, a derivative of E. coli W3110, for expressionof the Kunitz domain inhibitors. Overnight saturated cultures wereinoculated (1%) into 250 ml of low phosphate minimal media (Chang, C. N.et al., Gene 55: 189-196 1987!) containing 50 μg/ml ampicillin and grownfor 20 h at 37° C. Inhibitors were secreted into the periplasm by virtueof the stII signal sequence and eventually leaked into the media. Cellsand debris were removed by centrifugation (8000×g, 10 min); thesupernatant was adjusted to pH 7.5-8.5 with 1 M NaOH and then loadedonto a 1 ml trypsin-Affigel 10 affinity column which was preparedaccording to the manufacturer's recommendations. The column was washedwith 100 mM Tris pH 8, 100 mM NaCl, and 20 mM CaCl₂ and inhibitors wereeluted with 4 ml of 10 mM HCl, 0.5 M KCl. The inhibitors were furtherpurified using C18 reverse phase HPLC (250×4.6 mm, VYDAC); they wereloaded in 0.1% trifluoroacetic acid and eluted with a CH₃ CN gradientfrom 5 to 40 % at 1 ml/min. Elution profiles were monitored at both A₂₁₄and A₂₈₀. A single well resolved peak was detected for each inhibitorbetween 30 to 35 % CH₃ CN. Inhibitor sequences were verified for theproper mass using a Sciex API 3 mass spectrometer equipped with anarticulated electrospray source for mass analysis. Multiply charged ionsof horse myoglobin (MW=16,951 Da) were used for instrument calibration.

Example 3 Determination of Equilibrium Dissociation Constants

Enzyme inhibition assays were conducted in a microtiter format andabsorbance changes were monitored on an SLT EAR340AT plate readercontrolled by a Macintosh SE computer equipped with BiometallicsDeltaSoftII software. Nonlinear regression analysis was carried outusing KaleidaGraph v3.01 (Synergy Software).

Inhibitor stocks were diluted in the range of 5-2000 nM; concentrationswere accurately determined by titration with trypsin that had beenactive site-titrated using 4-methylumbelliferyl p-guanidinobenzoate(Jameson, G. W. et al., Biochem. J. 131: 107-117 1973!). After a 1 hincubation of 80 nM trypsin plus an aliquot of diluted inhibitor in 50mM Tris, pH 8.0, 100 mM NaCl, 10 mM CaCl₂, and 0.05 % Triton X-100 atroom temperature, 20 μl of 5 mM N.sup.α-benzoyl-L-argininep-nitroanilide was added to a total volume of 150 μl.The change in absorbance at 405 nm was then monitored. Theconcentrations determined assumed a 1:1 stoichiometry of inhibitor withtrypsin.

Assays to test the activity of APPI, TF7I-C, and other mutants againstcoagulation proteases were conducted using the following format.Aliquots (25 μL) from each well of a microtiter plate containing theserially diluted inhibitors were transferred into new microtiter plates,each containing a different protease (100 μL) in the appropriate buffer.The proteases tested (protease concentration, buffer, substrate) wereFVIIa (10 nM, Buffer A, 0.7 mM S2366), FXIa (1.0 nM, Buffer B containing1 mg/ml BSA, 0.7 mM S2366), plasma kallikrein (3.5 nM, Buffer B, 0.5 mMS2302), and plasmin (15 nM, Buffer B, 1 mM S2251). Buffer A contains 50mM Tris, pH 7.5, 100 mM NaCl, 10 mM CaCl₂, 0.5% BSA, 60 nM TF₁₋₂₄₃, and1 mM CHAPS (Sigma). Buffer B contains 50 mM Tris, pH 7.5, 100 mM NaCl, 2mM CaCl₂ and 0.005% Triton X-100.

After incubation of the substrate/inhibitor mixes at room temperaturefor 1-3 h, the appropriate substrate (20 μL) was added, and theabsorbance at 405 nm was monitored. Controls lacking inhibitor andenzyme were assayed to measure the uninhibited and substrate hydrolysisrates, respectively. Plots of the fractional rate versus inhibitorconcentration were fit by nonlinear regression analysis to equation 1and apparent equilibrium dissociation constants (K_(i) ^(*)) weredetermined. The concentrations of FXa, FXIIa, and kallikrein were activesite titrated with a quantitated sample of ecotin, a reversibletight-binding inhibitor of these enzymes from E. coli, which wasoverexpressed and purified as previously described (U.S. patentapplication Ser. No. 08/121004, filed Sep. 14, 1993). The concentrationsof FVIIa, FXIa, and kallikrein were active site titrated using aquantitated sample of TF7I-C. Both TF7I-C and ecotin were quantitatedusing active site titrated trypsin. The concentrations of FVIIa, FXa,FXIa, FXIIa and kallikrein agreed well (±10 %) with the manufacturers'specifications (data not shown). The concentrations of activated proteinC, thrombin and plasmin were based upon those of the supplier.

Results

The apparent K_(i) ^(*) values of wild type APPI, TF7I-C, as well astwenty-nine other mutant inhibitors for TF-FVIIa, and in some casesFXIa, Kallikrein, and Plasmin were determined. The amino acid sequencesof APPI, TF7I-C and the other mutant inhibitors are described in FIG. 4.The sequences of the mutant inhibitors I-18, I-49, I-14, I-16, II-4,II-3, II-6, III-27, III-30, TF7I-VY, TF7I-LY, TF7I-WY, TF7I-PG, IV-47C,IV-54C, IV-31B, IV-49C, IV-50C, IV-57C, IV-51C, IV-35B, IV-58C, IV-48C,IV-46C, IV-55C, IV-32B, IV-36B, IV-40B and 53b as well as TF7I-C are allbased on the wild type APPI sequence which is represented by:

R₁ -Xaa₁₁ -Xaa₁₂ -Xaa₁₃ -Xaa₁₄ -Xaa₁₅ -Xaa₁₆ -Xaa₁₇ -Xaa₁₈ -Xaa₁₉ -R₂Xaa₃₄ -R₃ -Xaa₃₈ -Xaa₃₉ R₄

In each case R₁ has the sequence:

Val-Arg-Glu-Val-Cys-Ser-Glu-Gln-Ala-Glu (SEQ ID NO: 6)

R₂ has the sequence:

Arg-Trp-Tyr-Phe-Asp-Val-Thr-Glu-Gly-Lys-Cys-Ala-Pro-Phe (SEQ ID NO: 14)

R₃ has the sequence:

Tyr-Gly-Gly; and

R₄ has the sequence:

Gly-Asn-Arg-Asn-Asn-Phe-Asp-Thr-Glu-Glu-Tyr-Cys-Ala-Ala-Val-Cys-Gly-Ser-Ala(SEQ ID NO: 23) or

Gly-Asn-Arg-Asn-Asn-Phe-Asp-Thr-Glu-Glu-Tyr-Cys-Met-Ala-Val-Cys-Gly-Ser-Ala(SEQ ID NO: 24).

Therefore, the sequences of the mutant inhibitors tested in this Exampleare:

I-18 R₁ -Pro-Gly-Val-Cys-Arg-Ala-Leu-Ile-Leu-R₂ -Phe-R₃ -Cys-Gly-R₄ (SEQID NO: 43)

I-49 R₁ -Pro-Gly-Trp-Cys-Arg-Ala-Leu-Ile-Lue-R₂ -Phe-R₃ -Cys-Gly-R₄ (SEQID NO: 44)

I-14 R₁ -Pro-Gly-Phe-Cys-Arg-Ala-Leu-Ile-Leu-R₂ -Phe-R₃ -Cys-Gly-R₄ (SEQID NO: 45)

I-16 R₁ -Gly-Gly-Trp-Cys-Arg-Ala-Leu-Ile-Leu-R₂ -Phe-R₃ -Cys-Gly-R₄ (SEQID NO: 46)

where R₄ is the sequence identified by SEQ ID NO: 24, and

II-4 R₁ -Pro-Gly-Pro-Cys-Arg-Ala-Met-Ile-Ser-R₂ -Phe-R₃ -Cys-Tyr-R₄ (SEQID NO: 47)

II-3 R₁ -Pro-Gly-Trp-Cys-Arg-Ala-Met-Ile-Ser-R₂ -Ile-R₃ -Cys-Gly-R₄ (SEQID NO: 48)

II-6 R₁ -Pro-Gly-Pro-Cys-Lys-Ala-Met-Ile-Ser-R₂ -Ile-R₃ -Cys-Trp-R₄ (SEQID NO: 49)

III-27 R₁ -Thr-Gly-Pro-Cys-Arg-Ala-Leu-Ile-Ser-R₂ -Trp-R₃ -Cys-Gly-R₄(SEQ ID NO: 50)

III-30 R₁ -Thr-Gly-Pro-Cys-Arg-Ala-Leu-Ile-Ser-R₂ -Tyr-R₃ -Cys-Gly-R₄(SEQ ID NO: 51)

TF7I-VY R₁ -Pro-Gly-Val-Cys-Arg-Ala-Leu-Ile-Leu-R₂ -Phe-R₃ -Cys-Tyr-R₄(SEQ ID NO: 52)

TF7I-LY R₁ -Pro-Gly-Leu-Cys-Arg-Ala-Leu-Ile-Leu-R₂ -Phe-R₃ -Cys-Tyr-R₄(SEQ ID NO: 53)

TF7I-WY R₁ -Pro-Gly-Trp-Cys-Arg-Ala-Leu-Ile-Leu-R₂ -Phe-R₃ -Cys-Tyr-R₄(SEQ ID NO: 54)

TF7I-PG R₁ -Pro-Gly-Pro-Cys-Arg-Ala-Leu-Ile-Leu-R₂ -Phe-R₃ -Cys-Gly-R₄(SEQ ID NO: 55)

IV-47C R₁ -Pro-Gly-Pro-Cys-Arg-Ala-Met-Met-Lys-R₂ -Ile-R₃ -Cys-His-R₄(SEQ ID NO: 56)

IV-54C R₁ -Pro-Gly-Pro-Cys-Arg-Ala-Leu-Met-Lys-R₂ -Val-R₃ -Cys-Tyr-R₄(SEQ ID NO: 57)

IV-31B R₁ -Pro-Gly-Pro-Cys-Arg-Ala-Leu-Met-Lys-R₂ -Val-R₃ -Cys-Phe-R₄(SEQ ID NO: 58)

IV-49C R₁ -Pro-Gly-Pro-Cys-Arg-Ala-Met-Met-Lys-R₂ -Ile-R₃ -Cys-Tyr-R₄(SEQ ID NO: 59)

IV-50C R₁ -Pro-Gly-Pro-Cys-Arg-Ala-Met-Tyr-Lys-R₂ -Ile-R₃ -Cys-Tyr-R₄(SEQ ID NO: 60)

IV-57C R₁ -Pro-Gly-Val-Cys-Arg-Ala-Met-Met-Lys-R₂ -Ile-R₃ -Cys-Gly-R₄(SEQ ID NO: 61)

IV-51C R₁ -Pro-Gly-Pro-Cys-Lys-Ala-Leu-Met-Arg-R₂ -Tyr-R₃ -Cys-Tyr-R₄(SEQ ID NO: 62)

IV-35B R₁ -Pro-Gly-Pro-Cys-Lys-Ala-Ile-Met-Lys-R₂ -Ile-R₃ -Cys-His-R₄(SEQ ID NO: 63)

IV-58C R₁ -Pro-Gly-Pro-Cys-Lys-Ala-Leu-Met-Lys-R₂ -Tyr-R₃ -Cys-His-R₄(SEQ ID NO: 64)

IV-48C R₁ -Pro-Gly-Pro-Cys-Lys-Ala-Leu-Met-Lys-R₂ -Trp-R₃ -Cys-Trp-R₄(SEQ ID NO: 65)

IV-46C R₁ -Pro-Gly-Pro-Cys-Lys-Ala-Met-Ile-Lys-R₂ -Leu-R₃ -Cys-Tyr-R₄(SEQ ID NO: 66)

IV-55C R₁ -Pro-Gly-Pro-Cys-Lys-Ala-Leu-Met-Lys-R₂ -Phe-R₃ -Cys-Tyr-R₄(SEQ ID NO: 67)

IV-32B R₁ -Pro-Gly-Pro-Cys-Lys-Ala-Leu-Met-Lys-R₂ -Tyr-R₃ -Cys-Tyr-R₄(SEQ ID NO: 68)

IV-36B R₁ -Pro-Gly-Pro-Cys-Lys-Ala-Leu-Met-Lys-R₂ -Val-R₃ -Cys-Tyr-R₄(SEQ ID NO: 69)

IV-40B R₁ -Pro-Gly-Ala-Cys-Lys-Ala-Met-Tyr-Lys-R₂ -Ile-R₃ -Cys-Gly-R₄(SEQ ID NO: 70)

53b R₁ -Pro-Gly-Pro-Gly-Arg-Ala-Leu-Ile-Leu-R₂ -Phe-R₃ -Ala-Tyr-R₄ (SEQID NO: 71)

and TF71-IC R₁ -Pro-Gly-Pro-Cys-Arg-Ala-Leu-Ile-Leu-R₂ -Phe-R₃-Cys-Tyr-R₄ (SEQ ID NO: 72)

where R₄ has the sequence identified by SEQ ID NO: 23.

The results of the K_(i) ^(*) determinations are presented in FIG. 4. Insome cases the K_(i) ^(*) was determined for FXIa, Kallikrein, andPlasmin as well as TF-FVIIa. As demonstrated, the substitutions with theexception of II-6, III-27, III-30 and IV-40B, resulted in more potentinhibitors of TF-FVIIa than wild type APPI.

Example 4 Coagulation Assays

Clotting times for normal human plasma were performed using the ACL 300Research Coagulation Analyzer. For the prothrombin time (PT) assays, theincubation time was set at 120 sec and acquisition time at 120 to 600sec depending on the expected outcome of the assay. Membranes from 293cells expressing full length TF (Paborsky, L. R. et al., Biochemistry28: 8072-8077 1989!) were premixed with CaCl₂. The sample (plasma andinhibitor) and reagent (CaCl₂ /TF) were automatically mixed togetherafter a 2 min incubation at 37 ° C. The clotting time was determined byoptical assessment. The total incubation time of inhibitor with plasmabefore addition of CaCl₂ /TF was ca. 5 min. Final concentrations were 2to 20 μM inhibitor, 3.7 nM TF (0.9 μg/ml by protein content), 22.5 mMCaCl₂, and 50% plasma in a total volume of 160 μL.

For the activated partial thromboplastin time (APTT) assays, theactivation time was set at 120 sec and acquisition time at 300 to 600sec depending on the expected outcome of the assay. Citrated normalhuman plasma and inhibitor were incubated together. The sample (plasmaand inhibitor) and activator (Instrumentation Laboratories Ellagicacid/Phospholipid mix Test Reagent) were automatically pipetted andincubated together for 2 min at 37° C., then CaCl₂ was added andclotting time determined by means of optical assessment. The totalincubation time of inhibitor with plasma was ca. 3 min before additionof activator, and 5 min before addition of CaCl₂. Final concentrationswere 0.01 to 15 μM inhibitor, 15.3 μg protein/ml 293 cell membranes, 8.3μM ellagic acid, 8.3 mM CaCl₂, and 33.3% plasma in a total volume of 162μL.

Results

The results are presented in Table I below.

                  TABLE I                                                         ______________________________________                                        In Vitro Clotting Times in Human Plasma (fold prolongation)                   for Selected Variants                                                         Inhibitor        PT    APTT                                                   ______________________________________                                        APPI             1.5   3.6                                                    TF7I-C           3.5   <7.0                                                   IV-32B           1.4   1.3                                                    IV-49C           2.5   2.0                                                    IV-54C           2.1   2.2                                                    ______________________________________                                    

The clotting time in the absence of any inhibitor for the PT was 30s andfor the APTT was 31s. The fold prolongation is reported at aconcentration of 40 μM inhibitor.

Example 5 Rabbit Deep Medial Injury Model

Male New Zealand white rabbits (˜4 kg) were anesthetized to surgicalanesthesia plane with an IM injection of Ketamine / Xylaxine. Therabbits were placed supine on a restraining board, warmed to 37° C., andthe neck and inner thigh area shaved. Teflon catheters were replaced ina marginal ear vein and femoral artery for drug delivery and samplecollection respectively. Prior to treatment, blood samples werecollected for coagulation tests (APTT and PT). Bleeding time wasassessed from a cut made in the cuticle portion of a hind limb nail.Incisions were made in the neck region and the entire left commoncarotid artery and its branches were surgically isolated. An ultrasonicflow probe (Transonics®) was placed on the common carotid approximately5 cm caudal to the common--internal bifurcation. After blood flowreached a stable baseline, drugs (saline or test compounds) weredelivered via the marginal ear vein. A deflated embolectomy catheter(Fogarty®, 3F) was then introduced into the lumen of the common via anincision in the lingual branch. Blood flow through the artery wasstopped briefly while the catheter was introduced and loosely securedwith 2-0 silk tie at the incision site. After the catheter was in placeand secure, blood flow was restored. The deflated balloon was advancedto within 2 mm of the flow probe and inflated with saline untilresistance of the vessel wall was felt. The catheter was pulled backwith a steady motion to the first branch and then deflated. Thisprocedure was repeated a total of six times for each experimentalanimal, after which the catheter was removed. The ballooning procedure,from first insertion to removal of the catheter took 3 to 5 minutes andresulted in an area of damage that was 1.5 to 2 cm in length. Over 40minutes, blood samples were taken for APTT and PT measurements, cuticlebleeding times were assessed and blood flow through the carotidmonitored. Duration of patency was defined as the total amount of time(maximum=40 minutes) that any measurable blood flow is detected in theartery. Patency rate refers to the percentage of animals tested who hadcarotid artery blood flow ≧5 minutes. At the end of the experiment therabbit was euthanized and the carotid artery removed and opened. If anythrombus was present, it was removed, blotted and the weight recorded.

Results

                  TABLE IX                                                        ______________________________________                                        Clotting Times in Rabbit Plasma (Fold Increase over Baseline @ 40 μM*      or 50 μM*                                                                                 APTT  PT                                                       ______________________________________                                        APPI*            4.9     1.4                                                  TF7I-C*          >10     6.9                                                  IV-32B.sup.+     4.4     1.2                                                  IV-49C.sup.+     6.9     4.4                                                  IV-54C.sup.+     5.4     3.5                                                  ______________________________________                                    

Samples of rabbit plasma were assayed using an MLA 800 coagulometer andDade reagents. Actin FS® was the activator in the APTT assay. RabbitThromboplastin with Ca++ was used for the PT assays; the rabbitthromboplastin was diluted two-fold. All mutant inhibitors testedprolonged clotting by at least one fold. TF7I-C showed the greatestinhibition of the surface mediated contact activation pathway, asmeasured by the activated partial thromboplastin time assay. A greaterthan 10 fold prolongation of the clotting time at 40μM was observed.

                  TABLE III                                                       ______________________________________                                        In Vivo: Rabbit Deep Medial Injury Model                                             Patency                                                                       (%)   Patency (N)                                                                             Duration (Min.)                                                                           Clot (mps)                                 ______________________________________                                        Saline Control                                                                         0        0/11     0         92.1 ± 6.6                            Heparin 1                                                                              67      6/9       23.3 ± 6.7                                                                           13.6 ± 4.7                            Heparin 2                                                                              100     10/10     35.2 ± 2.2                                                                           19.1 ± 8.5                            APPI     40      2/5       13.4 ± 8.5                                                                            49.5 ± 18.2                          TF7I-C   83      5/6       30.3 ± 6.7                                                                            19.4 ± 14.6                          IV-49C   83      5/6       33.3 ± 6.7                                                                           19.7 ± 9.6                            ______________________________________                                          Heparin was dosed as a 25 u/kg bolus followed by a continuous infusion o     1) 0.5 u/kg/min or 2) 1 u/kg/min. Other reagents were given as a 2 mg/kg      IV bolus. With the exception of the patency data, values are expressed as     means ±sem.                                                           

Compared to the saline control, Heparin and the mutant APPI inhibitorsTF7I-C and IV-49C significantly prolonged patency and reduced clot sizefor the time periods studied. For both TF7I-C and IV-49C the percentageof animals that remained patent for greater than or equal to 5 minuteswas 83% compared to only 40% for the wild type APPI.

Table 3 below describes the results of the cuticle bleeding time assayat 10 minutes after dosing.

                  TABLE IV                                                        ______________________________________                                        Cuticle Bleeding Times (Fold Increase over Pre Dose)                                        N    Fold Increase                                              ______________________________________                                        Saline Control  6      1.0 ± 0.1                                           Heparin 1       ND                                                            Heparin 2       5      1.7 ± 0.4                                           APPI            4      2.2 ± 0.6                                           TF7I-C          4      0.7 ± 0.1                                           IV-49C          6      1.0 ± 1.0                                           ______________________________________                                    

As demonstrated by the results of the cuticle bleeding time assay,mutant APPI inhibitors such as TF7I-C and IV-49C may have improvedsafety profiles compared to current anticoagulant drugs such as Heparin.These two mutant inhibitors did not induce prolonged bleeding comparedto the saline control after 10 minutes. * * * * * * * * * *

All references cited herein are expressly incorporated by reference.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 72                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8 amino acids                                                     (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       AspIleCysLysLeuProLysAsp                                                      158                                                                           (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       ProGlyPheAlaLysAlaIleIleArg                                                   159                                                                           (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       ThrGlyLeuCysLysAlaTyrIleArg                                                   159                                                                           (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       ThrGlyLeuCysLysAlaArgIleArg                                                   159                                                                           (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       AlaGlyAlaAlaLysAlaLeuLeuAla                                                   159                                                                           (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino acids                                                    (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       ValArgGluValCysSerGluGlnAlaGlu                                                1510                                                                          (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino acids                                                    (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       MetHisSerPheCysAlaPheLysAlaAsp                                                1510                                                                          (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino acids                                                    (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       LysProAspPheCysPheLeuGluGluAsp                                                1510                                                                          (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino acids                                                    (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       GlyProSerTrpCysLeuThrProAlaAsp                                                1510                                                                          (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino acids                                                    (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      LysGluAspSerCysGlnLeuGlyTyrSer                                                1510                                                                          (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino acids                                                    (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      ThrValAlaAlaCysAsnLeuProIleVal                                                1510                                                                          (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino acids                                                    (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      LeuProAsnValCysAlaPheProMetGlu                                                1510                                                                          (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino acids                                                    (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      ArgProAspPheCysLeuGluProProTyr                                                1510                                                                          (2) INFORMATION FOR SEQ ID NO:14:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 amino acids                                                    (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                      ArgTrpTyrPheAspValThrGluGlyLysCysAlaProPhe                                    151014                                                                        (2) INFORMATION FOR SEQ ID NO:15:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 amino acids                                                    (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                      ArgPhePhePheAsnIlePheThrArgGlnCysGluGluPhe                                    151014                                                                        (2) INFORMATION FOR SEQ ID NO:16:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 amino acids                                                    (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                      ArgTyrPheTyrAsnAsnGlnThrLysGlnCysGluArgPhe                                    151014                                                                        (2) INFORMATION FOR SEQ ID NO:17:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 amino acids                                                    (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                      ArgPheTyrTyrAsnSerValIleGlyLysCysArgProPhe                                    151014                                                                        (2) INFORMATION FOR SEQ ID NO:18:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 amino acids                                                    (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                      ArgTyrPheTyrAsnGlyThrSerMetAlaCysGluThrPhe                                    151014                                                                        (2) INFORMATION FOR SEQ ID NO:19:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 amino acids                                                    (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                      LeuTrpAlaPheAspAlaValLysGlyLysCysValLeuPhe                                    151014                                                                        (2) INFORMATION FOR SEQ ID NO:20:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 amino acids                                                    (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                      LysTrpTyrTyrAspProAsnThrLysSerCysAlaArgPhe                                    151014                                                                        (2) INFORMATION FOR SEQ ID NO:21:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 amino acids                                                    (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                      ArgTrpPhePheAsnPheGluThrGlyGluCysGluLeuPhe                                    151014                                                                        (2) INFORMATION FOR SEQ ID NO:22:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 amino acids                                                    (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                                      ArgTyrPheTyrAsnAlaLysAlaGlyLeuCysGlnThrPhe                                    151014                                                                        (2) INFORMATION FOR SEQ ID NO:23:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 amino acids                                                    (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                                      GlyAsnArgAsnAsnPheAspThrGluGluTyrCysAlaAlaVal                                 151015                                                                        CysGlySerAla                                                                  19                                                                            (2) INFORMATION FOR SEQ ID NO:24:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 amino acids                                                    (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                                      GlyAsnArgAsnAsnPheAspThrGluGluTyrCysMetAlaVal                                 151015                                                                        CysGlySerAla                                                                  19                                                                            (2) INFORMATION FOR SEQ ID NO:25:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 amino acids                                                    (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                                      GlyAsnGlnAsnArgPheGluSerLeuGluGluCysLysLysMet                                 151015                                                                        CysThrArgAsp                                                                  19                                                                            (2) INFORMATION FOR SEQ ID NO:26:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 amino acids                                                    (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                                      GlyAsnMetAsnAsnPheGluThrLeuGluGluCysLysAsnIle                                 151015                                                                        CysGluAspGly                                                                  19                                                                            (2) INFORMATION FOR SEQ ID NO:27:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 amino acids                                                    (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                                      GlyAsnGluAsnAsnPheThrSerLysGlnGluCysLeuArgAla                                 151015                                                                        CysLysLysGly                                                                  19                                                                            (2) INFORMATION FOR SEQ ID NO:28:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 amino acids                                                    (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:                                      GlyAsnGlyAsnAsnPheValThrGluLysGluCysLeuGlnThr                                 151015                                                                        CysArgThrVal                                                                  19                                                                            (2) INFORMATION FOR SEQ ID NO:29:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 amino acids                                                    (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:                                      GlyAsnGlyAsnLysPheTyrSerGluLysGluCysArgGluTyr                                 151015                                                                        CysGlyValPro                                                                  19                                                                            (2) INFORMATION FOR SEQ ID NO:30:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 amino acids                                                    (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:                                      GlyAsnGluAsnLysPheGlySerGlnLysGluCysGluLysVal                                 151015                                                                        CysAlaProVal                                                                  19                                                                            (2) INFORMATION FOR SEQ ID NO:31:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 amino acids                                                    (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:                                      GlyAsnSerAsnAsnPheLeuArgLysGluLysCysGluLysPhe                                 151015                                                                        CysLysPheThr                                                                  19                                                                            (2) INFORMATION FOR SEQ ID NO:32:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 amino acids                                                    (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:                                      AlaLysArgAsnAsnPheLysSerAlaGluAspCysMetArgThr                                 151015                                                                        CysGlyGlyAla                                                                  19                                                                            (2) INFORMATION FOR SEQ ID NO:33:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 58 amino acids                                                    (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:                                      ValArgGluValCysSerGluGlnAlaGluProGlyProCysArg                                 151015                                                                        AlaLeuIleLeuArgTrpTyrPheAspValThrGluGlyLysCys                                 202530                                                                        AlaProPhePheTyrGlyGlyCysTyrGlyAsnArgAsnAsnPhe                                 354045                                                                        AspThrGluGluTyrCysAlaAlaValCysGlySerAla                                       505558                                                                        (2) INFORMATION FOR SEQ ID NO:34:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 58 amino acids                                                    (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:                                      ValArgGluValCysSerGluGlnAlaGluThrGlyProCysArg                                 151015                                                                        AlaMetIleSerArgTrpTyrPheAspValThrGluGlyLysCys                                 202530                                                                        AlaProPhePheTyrGlyGlyCysGlyGlyAsnArgAsnAsnPhe                                 354045                                                                        AspThrGluGluTyrCysMetAlaValCysGlySerAla                                       505558                                                                        (2) INFORMATION FOR SEQ ID NO:35:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 58 amino acids                                                    (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:                                      MetHisSerPheCysAlaPheLysAlaAspAspGlyProCysLys                                 151015                                                                        AlaIleMetLysArgPhePhePheAsnIlePheThrArgGlnCys                                 202530                                                                        GluGluPheIleTyrGlyGlyCysGluGlyAsnGlnAsnArgPhe                                 354045                                                                        GluSerLeuGluGluCysLysLysMetCysThrArgAsp                                       505558                                                                        (2) INFORMATION FOR SEQ ID NO:36:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 58 amino acids                                                    (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:                                      LysProAspPheCysPheLeuGluGluAspProGlyIleCysArg                                 151015                                                                        GlyTyrIleThrArgTyrPheTyrAsnAsnGlnThrLysGlnCys                                 202530                                                                        GluArgPheLysTyrGlyGlyCysLeuGlyAsnMetAsnAsnPhe                                 354045                                                                        GluThrLeuGluGluCysLysAsnIleCysGluAspGly                                       505558                                                                        (2) INFORMATION FOR SEQ ID NO:37:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 58 amino acids                                                    (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:                                      GlyProSerTrpCysLeuThrProAlaAspArgGlyLeuCysArg                                 151015                                                                        AlaAsnGluAsnArgPheTyrTyrAsnSerValIleGlyLysCys                                 202530                                                                        ArgProPheLysTyrSerGlyCysGlyGlyAsnGluAsnAsnPhe                                 354045                                                                        ThrSerLysGlnGluCysLeuArgAlaCysLysLysGly                                       505558                                                                        (2) INFORMATION FOR SEQ ID NO:38:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 58 amino acids                                                    (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:                                      LysGluAspSerCysGlnLeuGlyTyrSerAlaGlyProCysMet                                 151015                                                                        GlyMetThrSerArgTyrPheTyrAsnGlyThrSerMetAlaCys                                 202530                                                                        GluThrPheGlnTyrGlyGlyCysMetGlyAsnGlyAsnAsnPhe                                 354045                                                                        ValThrGluLysGluCysLeuGlnThrCysArgThrVal                                       505558                                                                        (2) INFORMATION FOR SEQ ID NO:39:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 58 amino acids                                                    (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:                                      ThrValAlaAlaCysAsnLeuProIleValArgGlyProCysArg                                 151015                                                                        AlaPheIleGlnLeuTrpAlaPheAspAlaValLysGlyLysCys                                 202530                                                                        ValLeuPheProTyrGlyGlyCysGlnGlyAsnGlyAsnLysPhe                                 354045                                                                        TyrSerGluLysGluCysArgGluTyrCysGlyValPro                                       505558                                                                        (2) INFORMATION FOR SEQ ID NO:40:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 58 amino acids                                                    (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:                                      GluThrAspIleCysLysLeuProLysAspGluGlyThrCysArg                                 151015                                                                        AspPheIleLeuLysTrpTyrTyrAspProAsnThrLysSerCys                                 202530                                                                        AlaArgPheTrpTyrGlyGlyCysGlyGlyAsnGluAsnLysPhe                                 354045                                                                        GlySerGlnLysGluCysGluLysValCysAlaProVal                                       505558                                                                        (2) INFORMATION FOR SEQ ID NO:41:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 58 amino acids                                                    (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:                                      LeuProAsnValCysAlaPheProMetGluLysGlyProCysGln                                 151015                                                                        ThrTyrMetThrArgTrpPhePheAsnPheGluThrGlyGluCys                                 202530                                                                        GluLeuPheAlaTyrGlyGlyCysGlyGlyAsnSerAsnAsnPhe                                 354045                                                                        LeuArgLysGluLysCysGluLysPheCysLysPheThr                                       505558                                                                        (2) INFORMATION FOR SEQ ID NO:42:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 58 amino acids                                                    (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:                                      ArgProAspPheCysLeuGluProProTyrThrGlyProCysLys                                 151015                                                                        AlaArgIleIleArgTyrPheTyrAsnAlaLysAlaGlyLeuCys                                 202530                                                                        GlnThrPheValTyrGlyGlyCysArgAlaLysArgAsnAsnPhe                                 354045                                                                        LysSerAlaGluAspCysMetArgThrCysGlyGlyAla                                       505558                                                                        (2) INFORMATION FOR SEQ ID NO:43:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 58 amino acids                                                    (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:                                      ValArgGluValCysSerGluGlnAlaGluProGlyValCysArg                                 151015                                                                        AlaLeuIleLeuArgTrpTyrPheAspValThrGluGlyLysCys                                 202530                                                                        AlaProPhePheTyrGlyGlyCysGlyGlyAsnArgAsnAsnPhe                                 354045                                                                        AspThrGluGluTyrCysAlaAlaValCysGlySerAla                                       505558                                                                        (2) INFORMATION FOR SEQ ID NO:44:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 58 amino acids                                                    (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:                                      ValArgGluValCysSerGluGlnAlaGluProGlyTrpCysArg                                 151015                                                                        AlaLeuIleLeuArgTrpTyrPheAspValThrGluGlyLysCys                                 202530                                                                        AlaProPhePheTyrGlyGlyCysGlyGlyAsnArgAsnAsnPhe                                 354045                                                                        AspThrGluGluTyrCysAlaAlaValCysGlySerAla                                       505558                                                                        (2) INFORMATION FOR SEQ ID NO:45:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 58 amino acids                                                    (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:                                      ValArgGluValCysSerGluGlnAlaGluProGlyPheCysArg                                 151015                                                                        AlaLeuIleLeuArgTrpTyrPheAspValThrGluGlyLysCys                                 202530                                                                        AlaProPhePheTyrGlyGlyCysGlyGlyAsnArgAsnAsnPhe                                 354045                                                                        AspThrGluGluTyrCysAlaAlaValCysGlySerAla                                       505558                                                                        (2) INFORMATION FOR SEQ ID NO:46:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 58 amino acids                                                    (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:                                      ValArgGluValCysSerGluGlnAlaGluGlyGlyTrpCysArg                                 151015                                                                        AlaLeuIleLeuArgTrpTyrPheAspValThrGluGlyLysCys                                 202530                                                                        AlaProPhePheTyrGlyGlyCysGlyGlyAsnArgAsnAsnPhe                                 354045                                                                        AspThrGluGluTyrCysAlaAlaValCysGlySerAla                                       505558                                                                        (2) INFORMATION FOR SEQ ID NO:47:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 58 amino acids                                                    (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:                                      ValArgGluValCysSerGluGlnAlaGluProGlyProCysArg                                 151015                                                                        AlaMetIleSerArgTrpTyrPheAspValThrGluGlyLysCys                                 202530                                                                        AlaProPhePheTyrGlyGlyCysTyrGlyAsnArgAsnAsnPhe                                 354045                                                                        AspThrGluGluTyrCysAlaAlaValCysGlySerAla                                       505558                                                                        (2) INFORMATION FOR SEQ ID NO:48:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 58 amino acids                                                    (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:                                      ValArgGluValCysSerGluGlnAlaGluProGlyTrpCysArg                                 151015                                                                        AlaMetIleSerArgTrpTyrPheAspValThrGluGlyLysCys                                 202530                                                                        AlaProPheIleTyrGlyGlyCysGlyGlyAsnArgAsnAsnPhe                                 354045                                                                        AspThrGluGluTyrCysAlaAlaValCysGlySerAla                                       505558                                                                        (2) INFORMATION FOR SEQ ID NO:49:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 58 amino acids                                                    (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:49:                                      ValArgGluValCysSerGluGlnAlaGluProGlyProCysLys                                 151015                                                                        AlaMetIleSerArgTrpTyrPheAspValThrGluGlyLysCys                                 202530                                                                        AlaProPheIleTyrGlyGlyCysTrpGlyAsnArgAsnAsnPhe                                 354045                                                                        AspThrGluGluTyrCysAlaAlaValCysGlySerAla                                       505558                                                                        (2) INFORMATION FOR SEQ ID NO:50:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 58 amino acids                                                    (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:50:                                      ValArgGluValCysSerGluGlnAlaGluThrGlyProCysArg                                 151015                                                                        AlaLeuIleSerArgTrpTyrPheAspValThrGluGlyLysCys                                 202530                                                                        AlaProPheTrpTyrGlyGlyCysGlyGlyAsnArgAsnAsnPhe                                 354045                                                                        AspThrGluGluTyrCysAlaAlaValCysGlySerAla                                       505558                                                                        (2) INFORMATION FOR SEQ ID NO:51:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 58 amino acids                                                    (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:51:                                      ValArgGluValCysSerGluGlnAlaGluThrGlyProCysArg                                 151015                                                                        AlaLeuIleSerArgTrpTyrPheAspValThrGluGlyLysCys                                 202530                                                                        AlaProPheTyrTyrGlyGlyCysGlyGlyAsnArgAsnAsnPhe                                 354045                                                                        AspThrGluGluTyrCysAlaAlaValCysGlySerAla                                       505558                                                                        (2) INFORMATION FOR SEQ ID NO:52:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 58 amino acids                                                    (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:52:                                      ValArgGluValCysSerGluGlnAlaGluProGlyValCysArg                                 151015                                                                        AlaLeuIleLeuArgTrpTyrPheAspValThrGluGlyLysCys                                 202530                                                                        AlaProPhePheTyrGlyGlyCysTyrGlyAsnArgAsnAsnPhe                                 354045                                                                        AspThrGluGluTyrCysAlaAlaValCysGlySerAla                                       505558                                                                        (2) INFORMATION FOR SEQ ID NO:53:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 58 amino acids                                                    (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:53:                                      ValArgGluValCysSerGluGlnAlaGluProGlyLeuCysArg                                 151015                                                                        AlaLeuIleSerArgTrpTyrPheAspValThrGluGlyLysCys                                 202530                                                                        AlaProPhePheTyrGlyGlyCysTyrGlyAsnArgAsnAsnPhe                                 354045                                                                        AspThrGluGluTyrCysAlaAlaValCysGlySerAla                                       505558                                                                        (2) INFORMATION FOR SEQ ID NO:54:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 58 amino acids                                                    (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:54:                                      ValArgGluValCysSerGluGlnAlaGluProGlyTrpCysArg                                 151015                                                                        AlaLeuIleLeuArgTrpTyrPheAspValThrGluGlyLysCys                                 202530                                                                        AlaProPhePheTyrGlyGlyCysTyrGlyAsnArgAsnAsnPhe                                 354045                                                                        AspThrGluGluTyrCysAlaAlaValCysGlySerAla                                       505558                                                                        (2) INFORMATION FOR SEQ ID NO:55:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 58 amino acids                                                    (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:55:                                      ValArgGluValCysSerGluGlnAlaGluProGlyProCysArg                                 151015                                                                        AlaLeuIleLeuArgTrpTyrPheAspValThrGluGlyLysCys                                 202530                                                                        AlaProPhePheTyrGlyGlyCysGlyGlyAsnArgAsnAsnPhe                                 354045                                                                        AspThrGluGluTyrCysAlaAlaValCysGlySerAla                                       505558                                                                        (2) INFORMATION FOR SEQ ID NO:56:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 58 amino acids                                                    (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:56:                                      ValArgGluValCysSerGluGlnAlaGluProGlyProCysArg                                 151015                                                                        AlaMetMetLysArgTrpTyrPheAspValThrGluGlyLysCys                                 202530                                                                        AlaProPheIleTyrGlyGlyCysHisGlyAsnArgAsnAsnPhe                                 354045                                                                        AspThrGluGluTyrCysAlaAlaValCysGlySerAla                                       505558                                                                        (2) INFORMATION FOR SEQ ID NO:57:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 58 amino acids                                                    (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:57:                                      ValArgGluValCysSerGluGlnAlaGluProGlyProCysArg                                 151015                                                                        AlaLeuMetLysArgTrpTyrPheAspValThrGluGlyLysCys                                 202530                                                                        AlaProPheValTyrGlyGlyCysTyrGlyAsnArgAsnAsnPhe                                 354045                                                                        AspThrGluGluTyrCysAlaAlaValCysGlySerAla                                       505558                                                                        (2) INFORMATION FOR SEQ ID NO:58:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 58 amino acids                                                    (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:58:                                      ValArgGluValCysSerGluGlnAlaGluProGlyProCysArg                                 151015                                                                        AlaLeuMetLysArgTrpTyrPheAspValThrGluGlyLysCys                                 202530                                                                        AlaProPheValTyrGlyGlyCysPheGlyAsnArgAsnAsnPhe                                 354045                                                                        AspThrGluGluTyrCysAlaAlaValCysGlySerAla                                       505558                                                                        (2) INFORMATION FOR SEQ ID NO:59:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 58 amino acids                                                    (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:59:                                      ValArgGluValCysSerGluGlnAlaGluProGlyProCysArg                                 151015                                                                        AlaLeuMetLysArgTrpTyrPheAspValThrGluGlyLysCys                                 202530                                                                        AlaProPheIleTyrGlyGlyCysTyrGlyAsnArgAsnAsnPhe                                 354045                                                                        AspThrGluGluTyrCysAlaAlaValCysGlySerAla                                       505558                                                                        (2) INFORMATION FOR SEQ ID NO:60:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 58 amino acids                                                    (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:60:                                      ValArgGluValCysSerGluGlnAlaGluProGlyProCysArg                                 151015                                                                        AlaMetTyrLysArgTrpTyrPheAspValThrGluGlyLysCys                                 202530                                                                        AlaProPheIleTyrGlyGlyCysTyrGlyAsnArgAsnAsnPhe                                 354045                                                                        AspThrGluGluTyrCysAlaAlaValCysGlySerAla                                       505558                                                                        (2) INFORMATION FOR SEQ ID NO:61:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 58 amino acids                                                    (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:61:                                      ValArgGluValCysSerGluGlnAlaGluProGlyValCysArg                                 151015                                                                        AlaMetMetLysArgTrpTyrPheAspValThrGluGlyLysCys                                 202530                                                                        AlaProPheIleTyrGlyGlyCysGlyGlyAsnArgAsnAsnPhe                                 354045                                                                        AspThrGluGluTyrCysAlaAlaValCysGlySerAla                                       505558                                                                        (2) INFORMATION FOR SEQ ID NO:62:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 58 amino acids                                                    (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:62:                                      ValArgGluValCysSerGluGlnAlaGluProGlyProCysLys                                 151015                                                                        AlaLeuMetArgArgTrpTyrPheAspValThrGluGlyLysCys                                 202530                                                                        AlaProPheTyrTyrGlyGlyCysTyrGlyAsnArgAsnAsnPhe                                 354045                                                                        AspThrGluGluTyrCysAlaAlaValCysGlySerAla                                       505558                                                                        (2) INFORMATION FOR SEQ ID NO:63:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 58 amino acids                                                    (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:63:                                      ValArgGluValCysSerGluGlnAlaGluProGlyProCysLys                                 151015                                                                        AlaIleMetLysArgTrpTyrPheAspValThrGluGlyLysCys                                 202530                                                                        AlaProPheIleTyrGlyGlyCysHisGlyAsnArgAsnAsnPhe                                 354045                                                                        AspThrGluGluTyrCysAlaAlaValCysGlySerAla                                       505558                                                                        (2) INFORMATION FOR SEQ ID NO:64:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 58 amino acids                                                    (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:64:                                      ValArgGluValCysSerGluGlnAlaGluProGlyProCysLys                                 151015                                                                        AlaLeuMetLysArgTrpTyrPheAspValThrGluGlyLysCys                                 202530                                                                        AlaProPheTyrTyrGlyGlyCysHisGlyAsnArgAsnAsnPhe                                 354045                                                                        AspThrGluGluTyrCysAlaAlaValCysGlySerAla                                       505558                                                                        (2) INFORMATION FOR SEQ ID NO:65:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 58 amino acids                                                    (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:65:                                      ValArgGluValCysSerGluGlnAlaGluProGlyProCysLys                                 151015                                                                        AlaLeuMetLysArgTrpTyrPheAspValThrGluGlyLysCys                                 202530                                                                        AlaProPheTrpTyrGlyGlyCysTrpGlyAsnArgAsnAsnPhe                                 354045                                                                        AspThrGluGluTyrCysAlaAlaValCysGlySerAla                                       505558                                                                        (2) INFORMATION FOR SEQ ID NO:66:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 58 amino acids                                                    (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:66:                                      ValArgGluValCysSerGluGlnAlaGluProGlyProCysLys                                 151015                                                                        AlaMetIleLysArgTrpTyrPheAspValThrGluGlyLysCys                                 202530                                                                        AlaProPheLeuTyrGlyGlyCysTyrGlyAsnArgAsnAsnPhe                                 354045                                                                        AspThrGluGluTyrCysAlaAlaValCysGlySerAla                                       505558                                                                        (2) INFORMATION FOR SEQ ID NO:67:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 58 amino acids                                                    (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:67:                                      ValArgGluValCysSerGluGlnAlaGluProGlyProCysLys                                 151015                                                                        AlaLeuMetLysArgTrpTyrPheAspValThrGluGlyLysCys                                 202530                                                                        AlaProPhePheTyrGlyGlyCysTyrGlyAsnArgAsnAsnPhe                                 354045                                                                        AspThrGluGluTyrCysAlaAlaValCysGlySerAla                                       505558                                                                        (2) INFORMATION FOR SEQ ID NO:68:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 58 amino acids                                                    (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:68:                                      ValArgGluValCysSerGluGlnAlaGluProGlyProCysLys                                 151015                                                                        AlaLeuMetLysArgTrpTyrPheAspValThrGluGlyLysCys                                 202530                                                                        AlaProPheTyrTyrGlyGlyCysTyrGlyAsnArgAsnAsnPhe                                 354045                                                                        AspThrGluGluTyrCysAlaAlaValCysGlySerAla                                       505558                                                                        (2) INFORMATION FOR SEQ ID NO:69:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 58 amino acids                                                    (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:69:                                      ValArgGluValCysSerGluGlnAlaGluProGlyProCysLys                                 151015                                                                        AlaLeuMetLysArgTrpTyrPheAspValThrGluGlyLysCys                                 202530                                                                        AlaProPheValTyrGlyGlyCysTyrGlyAsnArgAsnAsnPhe                                 354045                                                                        AspThrGluGluTyrCysAlaAlaValCysGlySerAla                                       505558                                                                        (2) INFORMATION FOR SEQ ID NO:70:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 58 amino acids                                                    (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:70:                                      ValArgGluValCysSerGluGlnAlaGluProGlyAlaCysLys                                 151015                                                                        AlaMetTyrLysArgTrpTyrPheAspValThrGluGlyLysCys                                 202530                                                                        AlaProPheIleTyrGlyGlyCysGlyGlyAsnArgAsnAsnPhe                                 354045                                                                        AspThrGluGluTyrCysAlaAlaValCysGlySerAla                                       505558                                                                        (2) INFORMATION FOR SEQ ID NO:71:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 58 amino acids                                                    (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:71:                                      ValArgGluValCysSerGluGlnAlaGluProGlyProGlyArg                                 151015                                                                        AlaLeuIleLeuArgTrpTyrPheAspValThrGluGlyLysCys                                 202530                                                                        AlaProPhePheTyrGlyGlyAlaTyrGlyAsnArgAsnAsnPhe                                 354045                                                                        AspThrGluGluTyrCysAlaAlaValCysGlySerAla                                       505558                                                                        (2) INFORMATION FOR SEQ ID NO:72:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 58 amino acids                                                    (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:72:                                      ValArgGluValCysSerGluGlnAlaGluProGlyProCysArg                                 151015                                                                        AlaLeuIleLeuArgTrpTyrPheAspValThrGluGlyLysCys                                 202530                                                                        AlaProPhePheTyrGlyGlyCysTyrGlyAsnArgAsnAsnPhe                                 354045                                                                        AspThrGluGluTyrCysAlaAlaValCysGlySerAla                                       505558                                                                        __________________________________________________________________________

What is claimed is:
 1. An isolated DNA molecule encoding a polypeptidecomprising a Kunitz-type serine protease inhibitor domain having anequilibrium dissociation constant of less than 100 nM for tissuefactor-Factor VIIa represented by Structural Formula (I):

    R.sub.1 -Xaa.sub.11 -Xaa.sub.12 -Xaa.sub.13 -Xaa.sub.14 -Xaa.sub.15 -Xaa.sub.16 -Xaa.sub.17 -Xaa.sub.18 -Xaa.sub.19 -R.sub.2 -Xaa.sub.34 R.sub.3 -Xaa.sub.38 -Xaa.sub.39 -R.sub.4                  (I)

where R₁ is a peptide having from 5 to 10 amino acid residues wherein atleast one residue is Cys; R₂ is a peptide having 14 amino residueswherein at least one residue is Cys; R₃ is a tripeptide; R₄ is a peptidehaving from 12 to 19 amino acid residues wherein at least one residue isCys; Xaa₁₁ is selected from the group consisting of Pro, Arg, Ala, Glu,Gly, and Thr; Xaa₁₂ is gly; Xaa₁₃ is selected from the group consistingof Pro, Leu, Trp, Val, Gly, Phe, His, Tyr, Ala, Ile, Glu, and Gln; Xaa₁₄is selected from the group consisting of Cys, Ala, Ser, Thr, and Gly;Xaa₁₅ is selected from the group consisting of Met, Arg, and Lys; Xaa₁₆is selected from the group consisting of Gly and Ala; Xaa₁₇ is selectedfrom the group consisting of met, Leu, Ile, Arg, Tyr, and Ser; Xaa₁₈ isselected from the group consisting of Ile, His, Leu, Met, Tyr, and Phe;Xaa₁₉ is selected from the group consisting of Leu, Arg, Ala, Lys, andIle; Xaa₃₄ is selected from the group consisting of phe, Ile, Ser, Leu,Tyr, Trp, and Val; Xaa₃₈ is selected from Cys, Ala, Ser, Thr, and Gly;and Xaa₃₉ is selected from the group Tyr, Gly, Trp, His, andPhe;provided R₁ is not Xaa₁ Asp-Ile-Cys-Lys-Leu-Pro-Lys-Asp (Seq ID NO:1), where Xaa₁ is His or 1-5 amino acid residues; and Xaa₁₁ throughXaa₁₉ are notPro-Gly-Phe-Ala-Lys-Ala-Ile-Ile-Arg (SEQ ID NO: 2);Thr-Gly-Leu-Cys-Lys-Ala-Tyr-Ile-Arg (SEQ ID NO: 3);Thr-Gly-Leu-Cys-Lys-Ala-Arg-Ile-Arg (SEQ ID NO: 4); andAla-Gly-Ala-Ala-Lys-Ala-Leu-Leu-Ala (SEQ ID NO: 5).
 2. The DNA moleculeof Claim 1 comprising an expression control sequence operably linked tothe DNA molecule.
 3. An expression vector comprising the DNA molecule ofclaim 2 wherein the control sequence is recognized by a host celltransformed with the vector.
 4. The vector of claim 3 that is a plasmid.5. A host cell transformed with the plasmid of claim
 4. 6. A method forexpressing a DNA molecule encoding a serine protease inhibitor in a hostcell, comprising culturing the host cell of Claim 5 under conditionssuitable for expression of the inhibitor.
 7. The method of claim 6further comprising recovering the inhibitor from the culture medium. 8.The DNA molecule of claim 1 wherein R₁ is a 10 amino acid peptide, R₄ is19 amino acid peptide and amino acid residues 5, 14, 30, 38, 51 and 55of the Kunitz-type domain are Cys.
 9. The DNA molecule of claim 8whereinXaa₁₁ is Pro; Xaa₁₂ is Gly; Xaa₁₃ is selected from the groupconsisting of Pro, Trp, Val, and Leu; Xaa₁₄ is Cys; Xaa₁₅ is selectedfrom the group consisting of Arg and Lys; Xaa₁₆ is Ala; Xaa₁₇ isselected from the group consisting of Leu, and Met; Xaa₁₈ is selectedfrom the group consisting of Met, and Ile; Xaa₁₉ is selected from thegroup consisting of Lys, Arg and Leu; Xaa₃₄ is selected from the groupconsisting of Phe, Val, Ile, Tyr; Xaa₃₉ is selected from the groupconsisting of Tyr, Gly, and His.
 10. The DNA moplecule of claim 9whereinXaa₁₇ is Leu; Xaa₁₈ is Met; and Xaa₁₉ is selected from the groupconsisting of Lys and Arg.
 11. The DNA molecule of claim 9 whereinXaa₁₇is Leu; Xaa₁₈ is Met; and Xaa₁₉ is Leu.
 12. The DNA molecule of claim 10wherein R₁ is selected from the group consistingof:Val-Arg-Glu-Val-Cys-Ser-Glu-Gln-Ala-Glu (SEQ ID NO: 6);Met-His-Ser-Phe-Cys-Ala-Phe-Lys-Ala-Asp (SEQ ID NO: 7);Lys-Pro-Asp-Phe-Cys-Phe-Leu-Glu-Glu-Asp (SEQ ID NO: 8);Gly-Pro-Ser-Trp-Cys-Leu-Thr-Pro-Ala-Asp (SEQ ID NO: 9);Lys-Glu-Asp-Ser-Cys-Gln-Leu-Gly-Tyr-Ser (SEQ ID NO: 10);Thr-Val-Ala-Ala-Cys-Asn-Leu-Pro-Ile-Val (SEQ ID NO: 11);Leu-Pro-Asn-Val-Cys-Ala-Phe-Pro-Met-Glu (SEQ ID NO: 12); andArg-Pro-Asp-Phe-Cys-Leu-Glu-Pro-Pro-Tyr (SEQ ID NO: 13);R₂ is selectedfrom the group consisting of:Arg-Trp-Tyr-Phe-Asp-Val-Thr-Glu-Gly-Lys-Cys-Ala-Pro-Phe (SEQ ID NO: 14);Arg-Phe-Phe-Phe-Asn-Ile-Phe-Thr-Agr-Gln-Cys-Glu-Glu-Phe (SEQ ID NO: 15);Arg-Tyr-Phe-Tyr-Asn-Asn-Gln-Thr-Lys-Gln-Cys-Glu-Arg-Phe (SEQ ID NO: 16);Arg-Phe-Tyr-Tyr-Asn-Ser-Val-Ile-Gly-Lys-Cys-Arg-Pro-Phe (SEQ ID NO: 17);Arg-Tyr-Phe-Tyr-Asn-Gly-Thr-Ser-Met-Ala-Cys-Glu-Thr-Phe (SEQ ID NO: 18);Leu-Trp-Ala-Phe-Asp-Ala-Val-Lys-Gly-Lys-Cys-Val-Leu-Phe (SEQ ID NO: 19);Lys-Trp-Tyr-Tyr-Asp-Pro-Asn-Thr-Lys-Ser-Cys-Ala-Arg-Phe (SEQ ID NO: 20);Arg-Trp-Phe-Phe-Asn-Phe-Glu-Thr-Gly-Glu-Cys-Glu-Leu-Phe (SEQ ID NO: 21);Arg-Tyr-Phe-Tyr-Asn-Ala-Lys-Ala-Gly-Leu-Cys-Gln-Thr-Phe (SEQ ID NO:22);R₃ is selected from the group consisting of: Tyr-Gly-Gly; andTyr-Ser-Gly; andR₄ is selected from the group consisting of:Gly-Asn-Arg-Asn-Asn-Phe-Asp-Thr-Glu-Glu-Tyr-Cys-Ala-Ala-Val-Cys-Gly-Ser-Ala(SEQ ID NO: 23);Gly-Asn-Arg-Asn-Asn-Phe-Asp-Thr-Glu-Glu-Tyr-Cys-Met-Ala-Val-Cys-Gly-Ser-Ala(SEQ ID NO: 24);Gly-Asn-Gln-Asn-Arg-Phe-Glu-Ser-Leu-Glu-Glu-Cys-Lys-Lys-Met-Cys-Thr-Arg-Asp(SEQ ID NO: 25);Gly-Asn-Met-Asn-Asn-Phe-Glu-Thr-Leu-Glu-Glu-Cys-Lys-Asn-Ile-Cys-Glu-Asp-Gly(SEQ ID NO: 26);Gly-Asn-Glu-Asn-Asn-Phe-Thr-Ser-Lys-Gln-Glu-Cys-Leu-Arg-Ala-Cys-Lys-Lys-Gly(SEQ ID NO: 27);Gly-Asn-Gly-Asn-Asn-Phe-Val-Thr-Glu-Lys-Glu-Cys-Leu-Gln-Thr-Cys-Arg-Thr-Val(SEQ ID NO: 28);Gly-Asn-Gly-Asn-Lys-Phe-Tyr-Ser-Glu-Lys-Glu-Cys-Arg-Glu-Tyr-Cys-Gly-Val-Pro(SEQ ID NO: 29);Gly-Asn-Glu-Asn-Lys-Phe-Gly-Ser-Gln-Lys-Glu-Cys-Glu-Lys-Val-Cys-Ala-Pro-Val(SEQ ID NO: 30);Gly-Asn-Ser-Asn-Asn-Phe-Leu-Arg-Lys-Glu-Lys-Cys-Glu-Lys-Phe-Cys-Lys-Phe-Thr(SEQ ID NO: 31); andAla-Lys-Arg-Asn-Asn-Phe-Lys-Ser-Ala-Glu-Asp-Cys-Met-Arg-Thr-Cys-Gly-Gly-Ala(SEQ ID NO: 32).
 13. The DNA molecule of claim 11 wherein R₁ is selectedfrom the group consisting of:Val-Arg-Glu-Val-Cys-Ser-Glu-Gln-Ala-Glu(SEQ ID NO: 6); Met-His-Ser-Phe-Cys-Ala-Phe-Lys-Ala-Asp (SEQ ID NO: 7);Lys-Plo-Asp-Phe-Cys-Phe-Leu-Glu-Glu-Asp (SEQ ID NO: 8);Gly-Pro-Ser-Trp-Cys-Leu-Thr-Pro-Ala-Asp (SEQ ID NO: 9);Lys-Glu-Asp-Ser-Cys-Gln-Leu-Gly-Try-Ser (SEQ ID NO: 10);Thr-Val-Ala-Ala-Cys-Asn-Leu-Pro-Ile-Val (SEQ ID NO: 11);Leu-Pro-Asn-Val-Cys-Ala-Phe-Pro-Met-Glu (SEQ ID NO: 12); andArg-Pro-Asp-Phe-Cys-Leu-Glu-Pro-Pro-Tyr (SEQ ID NO: 13);R₂ is selectedfrom the consisting of:Arg-Trp-Tyr-Phe-Asp-Val-Thr-Glu-Gly-Lys-Cys-Ala-Pro-Phe (SEQ ID NO: 14);Arg-Phe-Phe-Phe-Asn-Ile-Phe-Thr-Agr-Gln-Cys-Glu-Glu-Phe (SEQ ID NO: 15);Arg-Tyr-Phe-Tyr-Asn-Asn-Gln-Thr-Lys-Gln-Cys-Glu-Arg-Phe (SEQ ID NO: 16);Arg-Phe-Tyr-Tyr-Asn-Ser-Val-Ile-Gly-Lys-Cys-Arg-Pro-Phe (SEQ ID NO: 17);Arg-Tyr-Phe-Thr-Asn-Gly-Thr-Ser-Met-Ala-Cys-Glu-Thr-Phe (SEQ ID NO: 18);Leu-Trp-Ala-Phe-Asp-Ala-Val-Lys-Gly-Lys-Cys-Val-Leu-Phe (SEQ ID NO: 19);Lys-Trp-Tyr-Tyr-Asp-Pro-Phe-Thr-Lys-Ser-Cys-Ala-Arg-Phe (SEQ ID NO: 20);Arg-Trp-Phe-Phe-Asn-Phe-Glu-Thr-Gly-Glu-Cys-Glu-Leu-Phe (SEQ ID NO: 21);and Arg-Tyr-Phe-Tyr-Asn-Ala-Lys-Ala-Gly-Leu-Cys-Gln-Thr-Phe (SEQ ID NO:22);R₃ is selected from the group consisting of: Tyr-Gly-Gly; andTyr-Ser-Gly; andR₄ is selected from the group consisting of:Gly-Asn-Arg-Asn-Asn-Phe-Asp-Thr-Glu-Glu-Tyr-Cys-Ala-Ala-Val-Cys-Gly-Ser-Ala(SEQ ID NO: 23);Gly-Asn-Arg-Asn-Asn-Phe-Asp-Thr-Glu-Glu-Tyr-Cys-Met-Ala-Val-Cys-Gly-Ser-Ala(SEQ ID NO: 24);Gly-Asn-Gln-Asn-Arg-Phe-Glu-Ser-Leu-Glu-Glu-Cys-Lys-Lys-Met-Cys-Thr-Arg-Asp(SEQ ID NO: 25);Gly-Asn-Met-Asn-Asn-Phe-Glu-Thr-Leu-Glu-Glu-Cys-Lys-Asn-Ile-Cys-Glu-Asp-Gle(SEQ ID NO: 26);Gly-Asn-Glu-Asn-Asn-Phe-Thr-Ser-Lys-Gln-Glu-Cys-Leu-Arg-Ala-Cys-Lys-Lys-Gly(SEQ ID NO: 27);Gly-Asn-Gly-Asn-Asn-Phe-Val-Thr-Glu-Lys-Glu-Cys-Leu-Gln-Thr-Cys-Arg-Thr-Val(SEQ ID NO: 28);Gly-Asn-Gly-Asn-Lys-Phe-Tyr-Ser-Glu-Lys-Glu-Cys-Arg-Glu-Tyr-Cys-Gly-Val-Pro(SEQ ID NO: 29);Glu-Asn-Glu-Asn-Lys-Phe-Gly-Ser-Gln-Lys-Glu-Cys-Glu-Lys-Val-Cys-Ala-Pro-Val(SEQ ID NO: 30);Gly-Asn-Ser-Asn-Asn-Phe-Leu-Arg-Lys-Glu-Lys-Cys-Glu-Lys-Phe-Cys-Lys-Phe-Thr(SEQ ID NO: 31); andAla-Lys-Arg-Asn-Asn-Phe-Lys-Ser-Ala-Glu-Asp-Cys-Met-Arg-Thr-Cys-Gly-Gly-Ala(SEQ ID NO: 32).
 14. The DNA moleccule of claim 12 where R₁ isVal-Arg-Glu-Val-Cys-Ser-Glu-Gln-Ala-Glu- (SEQ ID NO: 6), R₂isArg-Trp-Try-Phe-Asp-Val-Thr-Glu-Gly-Lys-Cys-Ala-Pro-Phe (SEQ ID NO:14) and R₄ isGly-Asn-Arg-Asn-Asn-Phe-Asp-Thr-Glu-Glu-Tyr-Cys-Ala-Ala-Val-Cys-Gly-Ser-Ala(SEQ ID NO: 23).
 15. The DNA molecule of claim 13 where R₁ isVal-Arg-Glu-Val-Cys-Ser-Glu-Gln-Ala-Glu (SEQ ID NO: 6), R₂ isArg-Trp-Try-Phe-ASp-Val-Thr-Glu-Gly-Lys-Cys-Ala-Pro-Phe (SEQ ID NO: 14)and R₄ isGly-Asn-Arg-Asn-Asn-Phe-Asp-Thr-Glu-Glu-Tyr-Cys-Ala-Ala-Val-Cys-Gly-Ser-Ala(SEQ ID NO: 23).
 16. The DNA molecule of claim 14 wherein amino acidresidues 12 and 37 of the Kunitz-type domain are Gly, amino acidresidues 33 and 45 of the Kunitz-type domain are Phe, amino acid residue35 of the Kunitz-type domain is Tyr and amino acid residue 43 of theKunitz-type domain is Asn.
 17. The DNA molecule of claim 15 whereinamino acid residues 12 and 37 of the Kunitz-type domain are Gly, aminoacid residues 33 and 45 of the Kunitz-type domain are Phe, amino acidresidue 35 of the Kunitz-type domain is Tyr and amino acid residue 43 ofthe Kunitz-type domain is Asn.